Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle.Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in  As cells grow and divide, a strict balance of a large variety of lipid species needs to be established and maintained to meet the metabolic demands to support growth in response to changes in environmental conditions. Sphingolipids (SLs)1–10–2/M (Gap-2/mitotic) progression of the cell cycle , in particular C20-PHS , which is encoded by LCB1/LCB2  and TSC3 (regulatory subunit) and which catalyzes the condensation of serine and palmitoyl-CoA to LCBs; these are further metabolized to ceramides and complex SLs  SLs . The yeaplex SLs –41. SPT lex SLs –, 43. TheMDL1/2/3 . Single c groups –48. In y groups –. Phospho groups –, 50. SL  groups – Fig. 1)SaccharomLarge scale studies have indicated a possible biochemical interaction between the Orm proteins and Swe1 kinase , and evi56–S. cerevisiae strains used in this study are listed in cdc28Y19F allele was introduced into the BY strain background in two steps. First the mutant CDC28 ORF was PCR-amplified from genomic DNA prepared from the a cdc28Y19F TRP1 GAL-SWE1mycMAT::URA3 strain (kind gift from the laboratory of Daniel Lew) using primers 5′-GACTAATGCATCATGGCTTATGTATTATACTTGCTTATGT-3′and 5′-ACTAATGCATGCTCCTAACGGTTGGTCCTTTGGAATACC-3′. The amplified fragment was cut with NsiI and inserted into the pSH47/NATR plasmid. This plasmid was used as a template for PCR amplification of the Y19Fcdc28 ORF along with the neighboring NatR selection marker using primers 5′-TTTTTATACAATACATATATATATATATATATATATATTTACAAGAAAAGACATGGAGGCCCAGAATACCC-3′ and 5′-GCTCCTAACGGTTGGTCCTTTGGAATACC-3′. The PCR product was chromosomally integrated into wild type, swe1, ypk1, and ypk2 strains, and transformants were selected for ClonNAT resistance, as described previously , the SWE1 gene was amplified as a BamHI-NotI fragment by PCR using genomic DNA as the template and primers 5′-GACTAGGATCCAGTTCTTTGGACGAGGATGAAGAG-3′ and 5′-GACTAGCGGCCGCTCATATAAAAAATTTTGGCTTAGGTCCAAA-3′. The PCR fragment was inserted into the respective restriction sites of plasmid pYEX4T-1 (Clontech) downstream of the copper-inducible CUP1 promoter. The GST-Ypk1 fusion under control of the CUP1 promoter was constructed by the same strategy using primers 5′-GACTAGGATCCTATTCTTGGAAGTCAAAGTTTAAGTTTG-3′ and 5′-GACTAGCGGCCGCCTATCTAATGCTTCTACCTTGCACC-3′.For constructing an ORM2  was amplified from yeast genomic DNA and ligated into the pRS316 vector. An NheI restriction site was introduced after the start codon of ORM2 by QuikChange mutagenesis (Agilent Technologies). A fragment of NheI/XhoI-flanked 3xFLAG-ORM2 obtained by PCR was used to replace the untagged ORM2 with an N-terminal 3xFLAG-tagged ORM2 (under the ORM2 promoter) by homologous recombination.For the generation of 3xFLAG-Orm2, a fragment of HIS3 gene at the designated ORF were selected on histidine drop-out medium. Solid media had the same composition plus 2% agar. Sporulation medium contained 0.25% yeast extract, 0.1% glucose, and 1% potassium acetate. To inhibit de novo synthesis of SLs -containing agar plates. The same amount of DMSO was added to the control plates. Growth was monitored after 3–4 days of incubation at 30 °C.Sublethal concentrations of the SPT inhibitor myriocin  were used to analyze the sensitivity of different yeast strains toward SPT inhibition. Yeast strains were cultured overnight at 30 °C in YNB minimal medium or YNB uracil drop-out medium, and 10 as ypk2ypk1 FLAG-Orm2 and as ypk2 swe1ypk1 FLAG-Orm2 were precultured for 72 h, harvested by centrifugation, and then released into fresh minimal media containing 670 ng/ml myriocin to induce phosphorylation of Orm2. After 30 min of myriocin treatment (0 min time point), cells were then additionally treated with 50 μm 3MB-PP1  Ypk1as kinase inhibitor for 30 min. Three A600 units of cells were harvested at the indicated time points by centrifugation at 4,000 rpm for 5 min at 4 °C. The cell pellets were resuspended in ice-cold 12.5% TCA and frozen at −80 °C overnight. The samples were thawed on ice, and the TCA was removed by centrifugation. The pellet was washed twice with ice-cold 80% acetone, air-dried, and resuspended in Laemmli solution  and loading dye and boiled for 5 min  and probed with anti-FLAG M2 peroxidase (HRP-conjugated) antibody (Sigma) and detected using ECL Western blotting substrate (Pierce).Yeast strains or 5 min . The proA600 units of the mutant strains swe1 and Y19Fcdc28 overexpressing GST-SWE1 were inoculated in minimal medium (−Ura) and cultivated to the logarithmic growth phase. Expression of GST-SWE1 was induced for 2 h by the addition of 50 μm copper sulfate. Cells were treated with myriocin (670 ng/ml) or PHS (15 μm) for 60 min. Three A600 units of cells were harvested, and proteins were precipitated as described above. Proteins were resolved on 8% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (Bio-Rad). Anti-GST antibody  was used to detect GST-Swe1. Horseradish peroxidase-conjugated anti-goat IgG (Chemicon) was the secondary antibody, and ECL Western blotting Substrate (PierceTM) was used for detection. Coomassie staining of the gel served as loading control.0.15 A600 units of cells were harvested from a logarithmically growing culture by centrifugation and washed with water. The pellet was resuspended in 2 m NH4OH and 2 ml of chloroform/methanol (1:2); C17-dihydrosphingosine internal standard was added (Sigma). Cells were lysed by vortexing at high speed for 3–4 min in the presence of zirconium beads. The supernatant was collected into fresh Pyrex tubes, and 1.5 ml of chloroform and 3 ml of 0.5 m NH4OH were added. After vortexing and a 5-min centrifugation at 4,000 rpm, the top layer was aspirated, and to the bottom layer another 1 ml of chloroform and 3 ml of 60 mm KCl were added. The top layer was again aspirated after centrifugation; the bottom layer was dried under a stream of nitrogen and resuspended in 100 μl of methanol, 190 mm triethylamine . 80 μl of extract were transferred to an HPLC vial containing 20 μl of AccQ reagent (Waters) and allowed to react for 30 min at room temperature. Aminophospholipids were deacylated by adding 5 μl of 1 m KOH in methanol and incubation at 37 °C for 30 min. The samples were neutralized by the addition of 5 μl of 1 m acetic acid in methanol. Insoluble materials were removed by brief centrifugation, and the supernatant was transferred to a new HPLC vial. Analysis was performed using an HP Series II 1090 liquid chromatograph with HP Chemstation coupled to an Agilent 1100 series fluorescence detector, as described  using a 100× numerical aperture 1.4 oil objective and differential interference contrast optics.swe1 and tslcb1 mutants show a negative genetic interaction, suggesting that the cell cycle checkpoint responds to alterations in sphingolipid metabolism . Thus, the absence of the morphogenesis checkpoint kinase Swe1 renders cells highly sensitive to reduced levels of sphingolipids. Swe1 asserts its checkpoint regulation by phosphorylating and inhibiting cyclin-dependent kinase Cdc28 at its tyrosine 19 residue; upon inhibition of Cdc28, the cell cycle is arrested/delayed until all of the conducive conditions are met , further supporting the notion that the cell cycle checkpoint activation is linked to the abundance of SLs. Conversely, these results suggest that the absence of the Swe1 kinase checkpoint or lack of phosphorylation of its downstream target Cdc28 leads to decreased levels of SLs and loss of viability upon SPT inhibition. Thus, Swe1-dependent phosphorylation/inhibition of Cdc28 appears to be required to attenuate cell cycle progression until a certain SL threshold level is achieved. There is no evidence, however, that Ccd28 directly regulates SL synthesis.Recent high-throughput studies indicated that tabolism , 53. If tabolism  would beild type A. Thus,  are met , 57, 59.myriocin A, furtheswe1 mutant could be a pleiotropic effect unlinked to SL metabolism. To exclude this possibility, we performed the growth tests on medium plates co-supplemented with myriocin and the sphingoid long-chain base, PHS, that bypasses the SPT reaction. Indeed, PHS supplementation rescued the swe1 and Y19Fcdc28 growth defect in the presence of myriocin, indicating that the levels of LCBs are growth-limiting in the mutant strains (B). Further support for this assumption comes from growth experiments of strains lacking the activating SPT subunit, Tsc3 .Swe1 is an important cell cycle checkpoint, so we were concerned that the myriocin-induced growth defect observed in the  strains B. Furtheit, Tsc3 : tsc3 mueostasis C.Y19Fcdc28 mutant cells, which escape regulation by Swe1. As shown in Fig. 3A, Swe1 overexpression in the swe1 mutant cells led to only a minor increase in myriocin resistance, presumably due to the counterbalancing effects of increasing myriocin resistance by derepressing SPT while at the same time activating the cell cycle checkpoint by Cdc28 phosphorylation. Indeed, overexpression of Swe1 in the Y19Fcdc28 mutant restored myriocin resistance back to wild type levels , indicating an activation of cellular SL production. Moreover, overexpression of SWE1 in wild-type cells results in an elongated cell phenotype  and an extended cell cycle delay/arrest due to prolonged inhibition of Cdc28 . Interestingly, the level of GST-tagged Swe1 protein was much lower when expressed from a plasmid in swe1 mutants as compared with the Y19Fcdc28 mutant, confirming previous studies that showed a feedback regulation of Swe1 abundance by Cdc28 activity , suggesting a regulation of Swe1 expression/abundance by the levels of SLs. In addition, PHS supplementation attenuated GST-Swe1 abundance in the Y19Fcdc28 mutant, further supporting previous observations that Swe1 responds to altered sphingolipid metabolism  were used as a positive control. Indeed, the total levels of LCBs were significantly lower in the swe1 mutant compared with wild type and similar to the ypk1 deletion mutant (A) ; PHS levels were significantly reduced in both swe1 and ypk1 mutants as compared with wild type (C). A closer analysis of the PHS species revealed that deletion of SWE1 drastically reduced the levels of C20-PHS in comparison with both the wild type and the ypk1 mutant (D). Previous studies showed that C20-PHS levels strongly respond to exposure of cells to heat stress  or by supplementation of cells with PHS. Thus, Ypk1 (and Ypk2) activity is essential to phosphorylate and inactivate the Orm1/2 proteins to sustain SPT activity. Based on the growth tests in the presence of myriocin and the suppressive effects of deleting both ORM genes, we speculated that Swe1 may also play a role in phosphorylating and inhibiting the Orm proteins, similar to the Ypk1 kinase. According to this model, lack of Orm1/2 phosphorylation by the absence of Swe1 increases inhibition of SPT activity, leading to reduced levels of SLs and rendering swe1 mutant cells highly sensitive to myriocin.Orm proteins are heavily phosphorylated by Ypk1 and, to some extent, also by Ypk2, which attenuates their inhibitory activity on SPT , and lacYPK1 and YPK2 is lethal . Upon inhibition of Ypk1 activity with 3-MB-PP1 in the as ypk2ypk1 strain, Orm2 phosphorylation was markedly reduced but not abolished (B). Phosphorylation of FLAG-tagged Orm2 was completely absent when, in addition, SWE1 was deleted in the as ypk2ypk1 strain upon 30-min treatment with the Ypk1as inhibitor (C). Taken together, these data strongly support the notion that the morphogenesis checkpoint kinase Swe1 plays a regulatory role in controlling SL metabolism, either by directly phosphorylating the negative SPT regulator Orm2 or by activating an as yet unidentified kinase other than Ypk1 or Ypk2 that inhibits Orm2. A possible contribution of the kinase Npr1, which is regulated via the nutrient-sensitive TORC1 pathway, on Orm2 phosphorylation under the conditions examined cannot be ruled out; Ypk1- and Npr1-mediated phosphorylation of Orm2 are independent of each other . We suspect that the different sensitivities toward SPT inhibition are due to the different relative impact of each kinase in regulating Orm phosphorylation and, therefore, SPT activity. Ypk1 kinase, as reported  renders yeast cells sensitive to SPT inhibition; however, the sublethal doses for each mutant differ significantly . The ypk effects A. We susreported , 86.in vivo, we investigated whether the Swe1-Orm and the Ypk1-Orm pathways for the regulation of SL synthesis were connected. Growth tests in the presence of myriocin (A) already indicated additive effects of inhibition, suggesting independent pathways. To test whether Swe1 kinase could compensate for the deletion of YPK1, SWE1 was overexpressed in ypk1 and Y19Fypk1 cdc28 mutant strains. As shown in Fig. 7B, the lack of viability in the presence of myriocin was rescued neither by SWE1 overexpression in the ypk1 nor by SWE1 overexpression in the Y19Fypk1 cdc28 mutant. The data suggest that Swe1 cannot compensate for the lack of Ypk1 in Orm phosphorylation . Similarly, overexpression of YPK1 did not suppress the myriocin sensitivity of the swe1 or Y19Fcdc28 mutants . In contrast, overexpression of SWE1 rescued the myriocin-induced growth defect in both swe1  and Y19Fcdc28 mutant strains. These data clearly indicate that the Swe1 and Ypk1 kinases regulate SPT activity by different pathways and cannot compensate, when overexpressed, for each other's absence. In addition, the myriocin sensitivity of Y19Fcdc28 mutants, which do not respond to inactivation by Swe1, indicates the requirement for a critical level of SLs for normal cell growth if the Swe1/Cdc28 checkpoint remains unchecked.Because Swe1 affected Orm2 phosphorylation myriocin A alreadyISC1-encoded inositol sphingolipid phospholipase C. The isc1 deletion mutant is sensitive to genotoxic stress-inducing agents, and growth arrest is mediated by activation of Swe1  a homeostatic level of de novo synthesized SLs has to be attained to proceed through the cell cycle, and (ii) one of the functions of the Swe1 kinase checkpoint is to ensure that an adequate amount of SLs is generated by the cells. Because Swe1 regulates a broad range of reactions, we confirmed that the increased myriocin sensitivity of Swe1-deficient mutants was indeed due to a lack of LCB synthesis; the addition of PHS, which bypasses the SPT reaction, fully restored growth of swe1 or Y19Fcdc28 mutants in the presence of the drug.Our study shows that absence of the Swe1-mediated cell cycle checkpoint results in decreased levels of SLs and increased sensitivity against the SPT inhibitor myriocin. A similar sensitivity to myriocin of the ORM1 and ORM2 suppresses the myriocin sensitivity of the swe1 mutant strain. Notably, overexpression of SWE1 only partially rescued the myriocin-induced growth defect of the swe1 deletion mutant due to its inhibitory phosphorylation of Cdc28, which is known to induce a G2/M arrest, an elongated cell phenotype, and decreased vegetative growth  ; howeverack loop . Total l0 (Most notably, Swe1 activity itself is regulated by SLs that are derived from precursors generated by the breakdown of triacylglycerols by the lipases, Tgl3 and Tgl4 , 89. The0 . This plS. D. K. and T. D. conceived and coordinated the study and wrote the paper. N. C. designed, performed, and analyzed experiments shown in Figs. 2


Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding.+/H+ exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner.Here, we identify the human NaThis work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2.The online version of this article (doi:10.1186/s12915-016-0252-7) contains supplementary material, which is available to authorized users. Extracellular signal-regulated kinase 2 (ERK2) is a member of the mitogen-activated protein kinase (MAPK) family of kinases activated in response to numerous growth factors and cytokines, leading to phosphorylation and functional regulation of downstream targets. ERK2 has been linked to more than 200 different substrates whose phosphorylation by ERK2 is orchestrated by coordination of signaling networks through common binding to so-called scaffold proteins . The defMAPKs are S/T kinases that interact with targets and regulators via two types of domains, D-domains and F-sites –11. D-do+/H+ exchanger 1  is a major regulator of pH and volume in essentially all cells studied. Furthermore, NHE1 is involved in the regulation of cell proliferation, survival, motility, and other essential physiological processes, and its dysregulation contributes importantly to major human malignancies, including cancer and cardiovascular diseases 706–721 (D1), [LVNEELKGKVLGLSR]732–746 (D2), and [LEQKINNYLTVPA]676–688 (D3) (listed in the order of stringency) ) and recombinantly produced human iaERK2 by NMR spectroscopy, which previously had provided insight into the transient structure and conserved regions of hNHE1cdt   was used15N,1H-HSQCs at 5, 10, 15, 20, and 25 °C. NMR samples of 400 μL of 100 μM or 200 μM 15N-labeled hNHE1cdt or variants were prepared in PBS buffer, 5 mM EDTA, 5 mM ATP, 15 mM MgCl2, 0.01 % (w/v) NaN3, 1 mM PMSF, 0.5 mM DSS, 2 mM DTT, 10 % (v/v) 99.96 % D2O, pH 7.0. A reference 15N,1H-HSQC spectrum was recorded before addition of kinase. Phosphorylation was started by addition of 10 μL of 0.1 mg/mL  unlabeled active ERK2 (proteinkinase.de), resulting in a molar excess of hNHE1cdt:ERK2 of 1,800:1. Phosphorylation was monitored from extraction of peak position and volumes from a series of 15N,1H-HSQCs. Peak intensities were normalized to unperturbed residues (Q815). Kinetics of S693, S771, T779, and S785 phosphorylation were extracted from non-linear least squares fittings of disappearing peaks of the unphosphorylated state and/or appearing peaks of the phosphorylated state, as well as reporting neighbours to single exponentials , S771 , T779 (V777 and T779), and S785 ). Kinetics of S723 and S726 require fitting to bi-exponentials due to their crosstalk. For this purpose peak intensities reporting on the disappearing unphosphorylated state, the appearing phosphorylated state, and both intermediates I1 and I2 of both S723 and S726 were fitted. For the comparison of the NHE1cdt variants, one peak was chosen for each site and each variant, i.e. S693, S723, S771P, V777 (reporting on T779), and S785. The disappearing peak of S723 reports on both, S723 and S726 phosphorylation. Fully phosphorylated 15N,13C-hNHE1cdt was assigned from standard 3D triple resonance NMR experiments as above.Time course experiments were run at 25 °C. Assignments were transferred to 25 °C by recording 3- [2, 95 % humidity in α-Minimum Essential Medium Eagle (Sigma) with 10 % fetal bovine serum, 1 % L-glutamine, 1 % penicillin/streptomycin (Gibco). Every 3–4 days, cells were passaged by gentle trypsination, and only passages 5–35 were used for experiments. WT and variant hNHE1 were expressed in AP-1 cells as in [AP-1 cells , which are CHO-derived cells with no endogenous NHE activity  and no r3- , 82, werUntransfected AP-1 cells or AP-1 cells expressing WT or variant hNHE1 were grown to ~ 80 % confluence in 10 cm Petri dishes, and incubated for 15 min in absence or presence of 100 ng/ml recombinant human EGF (Sigma). Cells were subsequently lysed and processed for immunoblotting as described below.Immunoblotting was carried out essentially as in . AntibodImmunofluorescence analysis was carried out essentially as in . Antibod488 for 1 h, and treated with DAPI to stain nuclei. Finally, coverslips were washed in Duolink Buffer A, mounted on object glass with mounting buffer, and sealed with nail polish. Imaging was carried out with an Olympus BX-61 epifluorescence microscope using cellSens Dimensions V1.6 software. Images were taken as z-stacks and z-projection images were created. Further image processing and quantification were carried out in ImageJ.Proximity ligation assay was carried out with the Duolink II Detection Reagents Red kit from Sigma Aldrich. AP-1 WT cells were seeded on coverslips the day before assaying. Cells were washed in ice-cold PBS, fixed in 4 % PFA for 20 min on ice, and washed in Duolink II Buffer A. Quenching was carried out in 0.1 M glycine for 15 min followed by permeabilization in 0.5 % Triton X-100. After permeabilization, cells were washed in Duolink II Buffer A and added O-link blocking solution for 30 min. Incubation with primary antibodies for 60 min in a humidity chamber at 37 °C was followed by 60 min incubation with PLA probes diluted 1:5 in Duolink II Antibody Diluent buffer at 37 °C. Coverslips were washed in Duolink II Buffer A and added ligation solution diluted 1:5 for 30 min at 37 °C, followed by wash in Duolink II Buffer A. Amplification solution diluted 1:5 was carried out for 100 min at 37 °C. After amplification, coverslips were washed in Duolink Buffer A, incubated with phalloidinn = 3, or as means ± standard error of the mean (SEM) as indicated. ANOVA with Tukey post-test, or Student’s t-test, as appropriate, were used to test for statistically significant differences, with p < 0.05 as the significance level.Data from mammalian cell culture on NHE1 function, immunofluorescence, and immunoblotting are shown as individual experiments representative of at least Data supporting the results of this article are available in the Additional files Additional file 1: Figure S1.(a) Variation of amide chemical shift by a pH difference of 0.2 units for hNHE1cdt. (b) Native PAGE of recombinantly expressed ERK2 reveals < 20 % auto-phosphorylation that is removed upon addition of HePTP. (PDF 1208 kb)pH effect on chemical shifts of the hNHE1cdt and ERK2 autoactivation and dephosphorylation by HePTP. Additional file 2: Figure S2.In vitro hNHE1cdt-ERK2 interaction. (a–b) Combined chemical shift perturbations Δδ and peak intensity ratios of WT hNHE1cdt by interaction with ERK2 fully dephosphorylated by HePTP. (c) Combined chemical shift perturbations Δδ of hNHE1cdt D3-AXA by iaERK2. (d–e) Combined chemical shift perturbations Δδ and peak intensity ratios of hNHE1cdt D1D2-(AXA)2 by iaERK2. (f–g) Combined chemical shift perturbations Δδ of hNHE1cdt F1-A and F2-AA by iaERK2. (h) Internally urea referenced secondary chemical shifts (ΔδC’) of hNHE1cdt WT. (PDF 399 kb)Additional file 3: Figure S3.(a) Size exclusion chromatography profiles of the hNHE1cdt WT (blue), iaERK2 (black), and a mixture of both (red). Subtraction of the individual runs from the mixture identifies a broad underlying peak with elution properties of the complex (grey). (b) Zoom from panel (a). Fractions taken for SDS-PAGE analysis (see panel (c)) are indicated by grey bars. (c) Coomassie-stained SDS-PAGE of fractions from individual runs, as indicated in panel (b). (d) Mean residual ellipticity CD spectra of hNHE1cdt WT, iaERK2, and mixture reveal no significant gain of secondary structure upon complex formation. (PDF 5632 kb)Analysis of the hNHE1cdt-iaERK2 complex by size exclusion chromatography and circular dichroism (CD). Additional file 4: Figure S4.(a) Peak behaviour for residues of the D3-domain, the F1-site, and the F2-site upon titration with iaERK2. Q815 is shown as a negative example. (b) Plotted peak intensity changes (top) and chemical shifts (bottom) for residues of the D3-domain, the F1-site, and the F2-site upon titration with iaERK2. The D3-domain has the highest apparent affinity, and single residue non-linear least squares fittings reveals a Kdapp of 35 ± 13 μM, 11 ± 2 μM, and 8 ± 1 μM for L684, T685, and V686, respectively. Global fitting results in a Kdapp of 16 ± 2 μM for the hNHE1 D3-domain. The affinities for the F1- and the F2-sites are lower (>50 μM). (PDF 304 kb)Affinity of the hNHE1cdt to iaERK2. Additional file 5: Figure S5.(a) Position of docking domains in the NHE1cdt relative to phosphorylation sites. (b) Effect of primary phosphosite mutations (S693A and T779A) on NHE1cdt phosphorylation kinetics by aERK2. Mutation on either site leads to increased rates at the other site potentially due to the absence of one sixth of high affinity phosphosites (intramolecular competition). Interestingly, the absence of T779 phosphorylation leads to slower rates at S771 and S785, which are close enough to sense the status of T779. (c) Effect of D-domain and F-site mutations on NHE1cdt phosphorylation kinetics by aERK2. The order of phosphorylation events is the same for all variants, yet the rates are modulated by the mutations. (PDF 468 kb)Effect of mutations on phosphorylation kinetics. Additional file 6: Figure S6.(a) Immunofluorescence images of AP-1 cells (hNHE1 D1D2-(AXA)2 and D1D2D3-(AXA)3) treated or not for 15 min with EGF (100 ng/ml). Merged images were zoomed to highlight the co-localization of ERK1/2 and NHE1 (white arrows). All other variants are shown in Figure (b) Representative line scans across membrane areas of images as in (a). (PDF 2105 kb)NHE1 co-localizes with ERK2. Additional file 7: Figure S7.(a–d) Intrinsic disorder was predicted by PONDR-FIT and DISOPRED 3.1 for (a) Tpr, (b) Nup50, (c) Nup153, and (d) Nup214, and the positions of FXF repeats were indicated by X. Notably, the great majority of FXF repeats localize to predicted disordered regions. (PDF 353 kb)Disorder prediction for Tpr and nuclear pore proteins. Additional file 8:Supplementary materials and methods. (DOCX 25 kb)Additional file 9: Figure S8.(a) Coomassie-stained SDS-PAGE (Bio-Rad) of hNHE1cdt WT, variants, and iaERK2. A low molecular weight marker (LMW) was used as standard . (b) Phosphorylation of the hNHE1cdt variants by aERK2 leads to an upward shift on SDS-PAGE towards apparent higher molecular weight relative to the unphosphorylated state. (PDF 1194 kb)Purity of protein preparations.


Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed. O-linked β-N-acetylglucosaminylation, and ubiquitylation , which is able to catalyze the phosphorylation of rpS6 at all sites, was the first kinase identified , whereas both S6K2 isoforms (p54 S6K2 and p56 S6K2) are primarily nuclear  signaling to control rpS6 phosphorylation. Thus, ERK can promote S6K activation by enhancing its phosphorylation at T421/S424 sites  at T34 . However, this long-lasting model has been challenged by subsequent studies showing that MEFs from the double mutant S6K1/2 as well as from the rpS6 knockin mouse lines exhibit normal TOP translation (Tang et al., in vivo in the striatum where the pharmacologically-induced rpS6 phosphorylation by d-amphetamine, haloperidol, or papaverine relies on the activation of the cAMP/PKA/DARPP-32 pathway (Biever et al., Despite these evidences, the involvement of rpS6 phosphorylation in the control of translation in the nervous system is still controversial. Indeed, several findings in hippocampal neurons and slices correlated increased rpS6 phosphorylation with enhanced global and TOP-encoded protein synthesis following different forms of synaptic plasticity (Kelleher et al., Another possibility is that rpS6 phosphorylation, within or outside the ribosome, exerts functions unrelated to mRNA translation, for example by interacting with other cellular proteins, which might become active or inactive upon the binding with rpS6. Indeed, co-immunoprecipitation studies suggest either a direct or indirect interaction of rpS6 with several extraribosomal proteins, including heat-shock protein 90 (Kim et al., Arabidopsis Thaliana proposes a role of rpS6 in rRNA synthesis and rDNA transcription via its interaction with the histone deacetylase AtHD2B (Kim et al., Surprisingly, extraribosomal functions have been attributed to several ribosomal proteins Wool, . For insSince the pioneering studies performed four decades ago, many progresses have been made regarding the identification of signaling events leading to rpS6 phosphorylation. Although rpS6 phosphorylation is still considered as a readout of mTORC1/S6K activity, caution should be taken with this assumption since other intracellular cascades largely contribute to the regulation of rpS6 phosphorylation, as exemplified with the PKA/DARPP-32/PP-1 pathway in the striatum. One should also keep in mind that the different sites of phosphorylation can be regulated independently in various brain areas or different cell-types within a same brain region. Although rpS6 phosphorylation has been and will remain a valuable hallmark of neuronal activity, understanding its biological role in the brain is undoubtedly one of the major challenges of the coming years.AB, EV, and EP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation  Acute lung injury (ALI) is characterised by increased lung vascular permeability, along with a reduced alveolar liquid clearance capacity; these changes typically develop in tandem and lead to progressive deterioration of lung function23Microtubules (MTs), a key component of the cytoskeleton, are responsible for regulating many processes, including cell migration and mitosis, as well as for maintaining cell shape and organelle transport6769Microtubule-associated protein 4 (MAP4) is primarily recognised as a cytosolic MT-binding protein that is ubiquitously expressed in non-neural cells and has an important role in microtubule dynamics, as indicated in previous studies by ourselves and other researchers111213141216In this study, we investigated the role of MAP4-MT binding and the role of p38/MAPK signalling in endothelial cells in mediating lung vascular leakage and ALI. We found that LPS or TNF-α led to endothelial hyperpermeability due to MT depolymerisation in HPMECs by activating p38/MAPK and inducing MAP4 phosphorylation. Thus, MAP4 may play a major role in the maintenance of vascular integrity and may provide a new potential therapeutic strategy for decreasing hyperpermeability in ALI.in vitro. The permeability of the endothelial monolayer was tested by measuring the influx of FITC-conjugated dextran and the transendothelial electrical resistance (TER) across cells  mutant, which mimicked the non-phosphorylated form, by changing S696, S768, and S787 to alanine. HA-tagged MAP4 (Ala) or CMV-null was overexpressed at comparable levels in HPMECs and examined by Western blot analysis . The resTo study whether the phosphorylation status of MAP4 influences MT structure, a MAP4 (Ala) mutant was transfected into cultured HPMECs . The effP38/MAPK was identified as the upstream kinase of MAP4 phosphorylation in hypoxic cardiomyocytes in our previous studyWe next tested whether the activation of p38/MAPK signalling was essential for MAP4 phosphorylation. MKK6 (Glu), a constitutively activated p38 kinase activator, was overexpressed in HPMECs to activate the p38/MAPK signalling pathway . ConversOur previous work demonstrated that MAP4 phosphorylation induces MT disassembly and endothelial barrier dysfunction after LPS or TNF-α stimulation, and we identified p38/MAPK as the upstream kinase of MAP4 phosphorylation. However, whether the p38/MAPK is the pivotal kinase that mediates MT disassembly and endothelial barrier dysfunction under inflammatory conditions must still be determined.We then observed the effect of p38/MAPK activation and inhibition on endothelial barrier function and MT dynamics in HPMECs. SB203580 (5 uM) was found largely to abolish the LPS- or TNF-α-induced endothelial hyperpermeability, which resulted in 1.16- and 1.20-fold reduction in dextran leakage, and 1.31- and 1.46-fold increase in TER compared with those for LPS and TNF-α, respectively, whereas the transduction with MKK6 (Glu) under normal conditions resulted in hyperpermeability  , suggestWe next evaluated the critical role of MAP4 phosphorylation in mediating the p38/MAPK regulation of MT dynamics and endothelial barrier function . In thisIn this study, we identified a novel role of MAP4 phosphorylation in endothelial cells in mediating the LPS- or TNF-α-induced increase in lung vascular permeability. We showed that LPS- or TNF-α induced MAP4 phosphorylation through activation of the p38/MAPK pathway in HPMECs, which led to MT disassembly and a parallel increase in vascular permeability, as indicated by the markedly increased dextran leakage and the decreased TER across the monolayer cells.Altered vascular permeability and subsequent pulmonary oedema are perhaps some of the most significant characteristics of ALI patients21109Members of the MAPs family include the neuronal proteins MAP2 and tau, which are primarily localized to the axon and the dendrites, respectively, and MAP4, the cytosolic MT-binding protein, which is present in all non-neuronal vertebrate cells19241211192628An important question arises as to which signalling pathway in lung endothelial cells is responsible for the MAP4 phosphorylation-dependent MT disassembly under inflammatory conditions. Our previous studies have indicated that p38/MAPK is the upstream kinase involved in MAP4 phosphorylation, as well as the p38/MAPK involved in the regulation of endothelial barrier function111617It is well known that LPS or TNF-α may induce endothelial apoptosis through a caspase-dependent pathway; this apoptosis could also lead to hyperpermeability in endothelial cells313210In summary, we conclude that p38/MAPK-induced MAP4 phosphorylation plays a critical role in the LPS- or TNF-α-induced MT disassembly and the subsequent vascular hyperpermeability. Our data suggest that MAP4 may be a useful target for the specific modulation of inflammation-induced hyperpermeability in ALI.Human pulmonary microvascular endothelial cells  were grown in 6-well culture plates and maintained for 72 hr in endothelial cell medium (ECM) with 5% foetal bovine serum , 1% Endothelial Cell Growth Supplement , and 1% penicillin/streptomycin (P/S) solution  before the LPS and TNF-α treatments. The p38/MAPK inhibitor SB203580 , taxol , or an irreversible and cell permeable inhibitor of caspase-3  was added to these cultures, which were incubated at 37°C for 1 hr before the LPS and TNF-α treatments under serum-free conditions. LPS , TNF-α , protein A/G-Sepharose , HA , rabbit polyclonal anti-MAP4 , mouse monoclonal anti-porin , rabbit polyclonal α-tubulin , α-tubulin , P38/MAPK , p-P38/MAPK , GAPDH-HRP , and p-MAP4 (S768)  were used. We raised the rabbit polyclonal antibody against p-MAP4  or p-MAP4  using the C-terminal 14 amino acids (PNKEPPP(pS)PEKKAK or KVAEKRT(pS)PSKPSSA and the respective non-phosphorylated peptides) conjugated to bovine serum albumin (BSA). The preparation and verification of the antibodies are described in the Primers were designed to generate the MAP4 point mutations S696G, S768G, and S787G or S696A, S768A, and S787A through PCR reactions using the QuikChange® Multi Site-Directed Mutagenesis Kit  . To eval5 cells/well) were plated in the Transwell chambers; the volumes of culture media in the upper and lower compartments were 100 and 600 μl; and the cells were grown for 4 days and replaced with serum-free medium for 1 hr before treatment. The permeability was measured by the addition of FITC-dextran  for 1 hr.Cells were grown on Transwell compartments. Transwell tracer experiments with fluorescent dextran2).The cellular barrier properties were assessed by measuring the TER across confluent HPMECs using Millicell-ERS Cells were grown on glass coverslips and cultured as described above. The cells were then fixed in 4% paraformaldehyde for 20 min, permeabilised with 0.1% Triton X-100 in PBS for 25 min, and blocked in 10% goat serum for 1.5 hr. To obtain the MT structure, rabbit anti-α-tubulin primary antibodies  were diluted with PBS, and the coverslips were incubated at 4°C overnight. The coverslips were washed in PBS and then incubated with goat anti-rabbit secondary antibodies conjugated to cyanine 3  for 1 hr at 37°C. The cells were imaged using confocal microscopy .2, and 20% glycerol. The cells were incubated with MTSB + 0.1% Triton X-100 + protease inhibitor cocktail  + phenylmethylsulphonyl fluoride  + phosphatase inhibitor cocktail . The polymerised and free tubulin fractions were quantified using Western blot analysis.The free and polymerised tubulin fractions were isolated using a method previously described by Putnam et al.The free and polymerised tubulin fractions prepared as described in the above section were probed using mouse anti-α-tubulin  antibody; the GAPDH-HRP  in the supernatant was chosen as the internal control for the free tubulin, and the mouse anti-Porin 31HL (Ab-2)  in the Triton X-100 insoluble fraction was chosen as the control for polymerised tubulin. The tubulin fractions and whole cells were stimulated and then lysed. The protein extracts were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and probed with specific antibodies as follows: p38 , p-P38 , MAP4 , p-MAP4  , and rabbit polyclonal antibody against p-MAP4  or p-MAP4 . As a loading control, GAPDH was probed and visualised. The immunocomplexes were visualised and quantified using an enhanced chemiluminescence detection kit  and horseradish peroxidase-conjugated secondary antibodies.To search for the protein interacting with MAP4 and tubulin, cells were lysed in RIPA buffer with a protease inhibitor tablets. α-tubulin (Santa Cruz Biotechnology) antibody was incubated with cell lysate for 6 h at 4°C, then the complexes precipitated with protein A/G-Sepharose  overnight at 4°C. The precipitates were washed 5 times with PBS at 0°C and separated by SDS/PAGE and probed by rabbit anti-MAP4  antibody using Western blotting.An In Situ Cell Death Detection Kit  was used for the apoptosis assay. Fifteen representative images of each group were randomly chosen using an Olympus microscope , and the mean fluorescence intensity was analysed using Image-Pro Plus 5.0 software. The experiments were repeated 3 times, and the mean values were calculated.P values < 0.05 were considered statistically significant.Statistical analysis of the data was performed using SPSS  13.0 software, and the significance was evaluated by one-way analysis of variance (ANOVA) followed by post hoc tests. Y.H. and B.C. supervised the work; L.L., J.H. and Y.H. designed the experiments with help from D.Z.; L.L., J.H., Q.Z., T.H. and X.L. performed the experiments; L.L., X.Y. and H.M. analysed the data; and L.L., J.H., Y.H. and B.C. co-wrote the manuscript. All authors discussed the results and commented on the manuscript.supplementary information


We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs.Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs  Subsequent opening of cyclic nucleotide-gated (CNG) channels and Ca2+-activated Cl- channels depolarizes the ORNs, which fire action potentials to transmit the signal to the olfactory central nervous system (CNS). In contrast, much less is known about the mechanisms through which odorants decrease the output of ORNs, a process referred to as odor-evoked inhibition.The process of odor detection and identification begins when odorants bind to olfactory receptors (ORs) expressed in the cilia of primary olfactory receptor neurons (ORNs) in the olfactory epithelium (OE) to activate signal transduction. Odorants can inhibit as well as excite ORNs thereby integrating their responses to complex odor mixtures Ache, . The canOdor-evoked inhibition at the level of ORN is often associated with competitive interaction between the cognate ligand and an antagonist, as was studied in detail with the rat I7 receptor .2 and 5% CO2 that contained (in mM): 120 NaCl, 25 NaHCO3, 5 KCl, 1.25 Na2HPO4, 1 MgSO4, 1 CaCl2, 10 glucose, 305 mOsm, pH7.4. After enzymatic digestion in papain (1 mg/mL) supplemented low-Ca (0.6 μM buffered with CaEGTA) ACSF at 37°C for 15 min, the tissue was gently washed with normal oxygenated ACSF and accurately triturated with a large bore fire polished glass transfer pipette. The resulting suspension was filtered through a 40 μm cell strainer (Fisher Scientific) and stored at 4°C with added TurboDNAse  to prevent cell clumping. An aliquot of the suspension was mixed with 10 μM Fluo-3/AM (AnaSpec) containing 0.04% Pluronic F127 and placed on a glass coverslip coated with 1 mg/mL concanavalin A (Sigma-Aldrich) in a recording chamber . The chamber was transferred to the stage of an inverted microscope  equipped with a 10× /0.5NA Fluar objective. To increase the sample size, in each experimental session the field of view was further expanded using a 0.63× reducer video tube (Diagnostic Instruments) between the microscope and the camera. Depending on the quality of the preparation, approximately 300 ORNs sensitive to IBMX/forskolin stimulation could be analyzed at once. The cells were illuminated at 500 nm  and the emitted light was collected at 530 nm  by a 12-bit cooled CCD camera . Both the illumination system  and image acquisition were controlled by Imaging Workbench six Software (INDEC BioSystems).Experiments were performed as described previously  and changes in fluorescence intensity within each ROI were analyzed and expressed as the peak fractional change in fluorescent light intensity F/F0 where F0 is the baseline fluorescence before odor application. Responses were detected and measured when the change in fluorescence intensity exceeded two standard deviations above the median noise level. For quantitative comparison, the peak amplitudes of the responses of different cells were normalized to the saturated responses elicited by application of a mixture of 100 μM IBMX (a phosphodiesterase inhibitor) and 10 μM forskolin (a selective agonist of adenylate cyclases) to robustly activate the cAMP signaling pathway. Application of IBMX/forskolin was also used to identify functional ORNs.Plasmids encoding the adenoviral backbone genes and the shuttle vector were provided by Dr.Jeffrey Martens (University of Florida) and viruses were prepared according to established protocols . The same optical configuration and illumination used to image dissociated cells was utilized for en face imaging of OE ectopically expressing PLCdelta1-PH:GFP or the calcium sensor GCaMP6f. Translocation was measured as an increase in global intra-knob cytoplasmic fluorescence upon application of the odor and presented as (F-Fo)/Fo ratio. The calcium response presented as an increase of GCaMP6f fluorescence emanating from the knob and underlying cell body. A standard eGFP filter cube BP490 nm/535 nm  was used to image both probes. Acquisition and illumination were controlled as described above.To observe translocation of the PIPen face imaging of freshly dissected OE was performed on an inverted confocal microscope Nikon Ti/A1R+ controlled by the NIS Elements 4.20 Software. Images were processed using ImageJ  in HEK293T cells. PI3K activation results in an accumulation of PIP3 and in turn recruitment of Btk-PH:GFP to the plasma membrane. Application of the PI3K inhibitor LY294002 reduces the PIP3 level in the membrane and Btk-PH:GFP translocates to the cytoplasm. PI3K-dependent changes in fluorescence were measured using the same optical configuration and illumination used to image dissociated ORNs and the PIP2 probe.A constitutively active form of the catalytic subunit p110gamma of PI3K fused to a membrane anchor motif CAAX,  was co-expressed with the PIP3-isobutyl-1-methylxanthine (IBMX) and 7β-acetoxy-8,13-epoxy-1α, 6β, 9α-trihydroxylabd-14-en-11-one (forskolin) were obtained from Sigma-Aldrich. LY294002 (Tocris), U73122, U73343 and edelfosine (Cayman). Reagents were dissolved in anhydrous DMSO and the stocks were kept at −20°C. Odorants were delivered as aqueous solutions prepared in freshly oxygenated ACSF. Octanol (OOL) and citral (CIT) were of the highest purity (Fisher Scientific) and were prepared as 0.5 M stock solutions in anhydrous DMSO. A complex odor mixture  was diluted 1:10 in DMSO and then mixed to the final reported dilution with ACSF. ACSF supplemented with 0.1% DMSO, the odorant carrier, served as the control solution. Odorant stocks were kept at −20°C and the final aqueous solutions were prepared on the day of the experiment.All data are expressed as mean ± SEM. All analyses were performed with pClampfit 10.2 (Molecular Devices), Microsoft Excel and SigmaPlot 10  Software.PLCG2f 5′ACTCTCTTCTCTCTCAGCCTCCG3′, PLCG2r 5′CAACAAATTCAAGACGACGGTTGTG3′, PLCE1f 5′TCCAAGTGCACGATGTCTCTCCAG3′, PLCE1r 5′GACTGGTAGCCCGCTGTGTCAC3′, PLCB3f 5′CCAGAACCGACAAGTACAGAGCC3′, PLCB3r 5′TCCCAGCTGCATGACCATTGC3′) and 10 μl of each reaction was analyzed by agarose gel electrophoresis.Odor-responsive dissociated ORNs identified by calcium imaging were collected directly into RT-PCR buffer and immediately frozen. Thawed cells were used for reverse transcription with an anchored oligo dT primer using a Verso cDNA kit (Thermo Fisher). To allow analysis of the expression of a number of genes, the cDNAs were pre-amplified with Primestar HS DNA polymerase (Takara) using a cocktail of gene-specific primers and a primer nested within the anchored-dT primer by incubating at 95°C for 2 min followed by 15 cycles of 95°C for 30 s, 50°C for 30 s 72°C for 1 min, then 72°C for 5 min. Samples were aliquoted and immediately frozen for later analysis. Beta actin primers were used to test for contamination with genomic DNA prior to analysis of PLC expression and olfactory marker protein (OMP) primers were used to confirm that the cells were canonical ORNs. PCR detection of the PLC isoforms was performed with gene specific primers  for a single cell RT-PCR analysis  applied for 5 s evoked a quick increase in fluorescence in some ORN knobs, directly reflecting translocation of the probe from the plasma membrane into the cytoplasm of the knobs of 12% of all tested ORNs  was measured before and after pre-treatment with a PI3K-specific blocker  and/or a PLC-specific blocker , and any change in the amplitude of the signal was measured. The basic logic behind these and the subsequent physiological experiments with native ORNs is that complex odors evoke a net response that reflects the excitation evoked by one or more components tempered by any inhibition evoked by another component of the mixture, and that pharmacologically blocking the inhibition, if any, results in a net increase in the response magnitude. In cases of extreme inhibition where there is no initial detectable excitatory response, this can result in a measurable response after blockade. For quantitative comparison, the peak amplitude of the responses of different ORNs were normalized to the saturated response elicited by application of a mixture of 100 μM IBMX and 10 μM forskolin  of the ORNs that were responsive to an odor mixture (H-100) increased their response following blockade of PI3K. Five of the 17 ORNs responded significantly to the mixture only when both enzymes were blocked, as in the original study  that increased their response to the odor mixture on blocking PI3K alone, however, showed no additional effect of blocking PLC Figure . In thesd Figure .In stark contrast to blocking PI3K alone, blocking PLC alone did not affect the response to the odor mixture in any of the cells tested, even in those in which blocking PI3K indicated there was inhibition to be relieved Figures . This waPreviously, we identified several inhibitory odorant pairs and showed that the increase in the strength of the response following PI3K blockade reflects a PI3K-dependent increase in the agonistic strength of the “antagonist”  correlates roughly with that of PI3K-based inhibition evoked by the same odor mixture . Translocation did not occur in response to a mixture of IBMX and forskolin, suggesting that it is evoked via a cAMP-independent pathway. While these findings are consistent with PLC activation in concert with PI3K in the context of odor-evoked inhibition, they are also compatible with recently described PLC-dependent odor-evoked excitatory calcium responses . Given that blockade of PLC alone is not sufficient to enhance odor-evoked excitation and that activation of PLC in concert with activation of PI3K is not an obligatory part of the pathway, our findings raise the question of whether the PLC effect is experimentally imposed. Both enzymes utilize a common substrate, PIP2 available as a substrate for PI3K, resulting in a proportionally larger increase in PIP3. This explanation is consistent with our observations that blocking PLC increases the odor-evoked response only when PI3K is blocked and that the consequence of blocking PLC alone is a decreased response. Since the ratio of inhibitory to excitatory odorants in H-100 for any given OR differs, we assume the level of inhibition, and therefore the demand on the pool of PIP2, differs across cells so that the effect of blocking PLC by itself would not necessarily result in a decrease in the response of every ORN. This is consistent with our finding that blocking PLC alone failed to reduce the odor-evoked response in a few cells that displayed PI3K-dependent inhibition. Thus, there is a reasonable explanation as to how pharmacological manipulation could impose an apparent contribution of PLC to inhibitory signaling in mammalian ORNs, although more targeted experimentation is required for confirmation.Conversely, blocking PLC could increase the pool of PIPOur findings are not to suggest that PLC is without functional significance in ORNs, just that it is not necessary for the inhibitory pathway investigated here. Indeed, odor-evoked PLC activity in the transduction zone is required for there to be a pharmacologically imposed effect on inhibitory signaling. Odorants have long been known to activate PLC in isolated ciliary membrane preparations  activating a PI3K-dependent pathway that opposes ACIII signaling by the excitatory odorant (OOL) in a ligand selective manner. Exogeneous PIPKU performed imaging of live tissue and transfected cultured cells, immunohistochemistry and performed data analysis. KU and EC collected cells and performed RT-PCR analysis. EC designed and performed single cell RT-PCR. All authors contributed to designing the study and writing the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The degree of hydrolysis after pepsin, 14.42%, and pancreatin, 30.65%, were noted. The peptides with the highest ACE inhibitory properties were separated using ion exchange chromatography on DEAE-cellulose. Thirteen peptides fractions were obtained but only four showed potential antihypertensive properties. The highest inhibitory activity was determined for the fraction F8 (IC50 = 0.0014 mg/mL). This fraction was separated on Sephadex G10 and two peptide fractions were obtained. The peptides fraction (B) with the highest ACE inhibitory activity (IC50 = 0.073 mg/mL) was identified by ESI-MS/MS. The sequences of ACE inhibitory peptides were GGSGNY, DLKLP, GSSDNR, MRDLK, and HNTPSR. Based on Lineweaver-Burk plots for the fraction B, the kinetic parameters as Km, Vmax, and Ki and mode of inhibition were determined. This fraction belongs to uncompetitive inhibitor of ACE activity. The seeds of pea are the source of precursor protein, which releases the ACE inhibitory peptides as a result of enzymatic hydrolysis.Pea seeds represent a valuable source of active compounds that may positively influence health. In this study, the pea globulins were digested  In recent years, food has become the subject of research not only as a source of energy and basic nutrients, which are essential for proper development and functioning of the body, but also as a source of bioactive compounds that may have the positive influence on health condition –3. The pOne of the most important parts of human homeostasis is the renin-angiotensin-aldosterone system (RAS) whose influence on the regulation and functioning of the circulatory system and water-electrolyte has been known for many years. This system is associated with the maintenance of normal blood volume, blood pressure stability, and proper amount of sodium ions . Disorde in vitro digestion.The aim of this study was the identification of potentially bioaccessible ACE inhibitory peptides obtained by digestion under simulated gastrointestinal conditions of pea globulins. Pea seeds are a valuable source of protein, minerals, carbohydrates, vitamins, or active compounds: peptides or antioxidants. To data, no study has conducted the investigation of primary structure of angiotensin converting enzyme inhibitors from pea globulins afterPisum sativum var. Bajka) were obtained from Company of Horticulture Seeds and Nursery in Ożarów Mazowiecki, Poland. HHL (hippuryl-L-histidyl-L-leucine), pepstatin A, PMSF (phenylmethanesulfonyl fluoride), α-amylase from hog pancreas , pepsin from porcine gastric mucosa , pancreatin from porcine pancreas , enalapril, bile extract, and TNBS  were purchased from Sigma-Aldrich Company, USA, and Amino Acid Standard from Pierce, USA. Any other chemicals were of analytical grade.Pea seeds  was added  and the mixture was stirred for 10 min at 37°C in darkness. For the gastric digestion, solution was adjusted to pH 2.5 with 1 M HCl and pepsin (250 U/mg) was added . Reaction was carried out for 2 h at 37°C. Solution was neutralized to pH 7.0 with 1 M NaOH and successively a mixture containing 0.7% solution of pancreatin and 2.5% solution of bile extract  was added . The incubation was carried out for 1 h at 37°C in darkness and reaction was stopped by heating at 100°C for 5 min.ik-Dziki  with sliHydrolysates were dialyzed with the dialysis sacks  against phosphate buffered saline (PBS) at the physiological concentration 1 : 4, v/v (simulated absorption process). The process was carried out in darkness for 1 h at 37°C. After this stage, the samples were concentrated tenfold using a vacuum evaporator; the parameters were set as follows: 40°C, pressure 0.8 mPa.Free amino groups content was determined by the trinitrobenzenesulfonic acid (TNBS) method using L-leucine as the standard . All ass in vitro digestion was estimated by determination of free amino groups by reaction with TNBS according to the method by Adler-Nissen [h are peptide bounds in the sample, htot are the total peptide bounds.The degree of hydrolysis (DH) afterr-Nissen . The totμL of peptide sample was added to 50 μL of 5 mM HHL solution and 50 μL of 3 mU/mL ACE (one unit of ACE activity was defined as an increase absorbance of 0.001 per minute at 390 nm). The reaction mixture was incubated at 37°C for 60 min. The reaction was terminated by adding 0.7 mL of the 0.1 M borate buffer with 0.2 M NaOH. The absorbance was measured at 390 nm and the ACE inhibition was determined as follows:A1 is the absorbance of sample with ACE and peptide inhibitor, A2 is the absorbance of sample with peptide inhibitor, without ACE, and A3 is absorbance of sample with ACE and without peptide inhibitor but with 0.1 M borate buffer pH 8.3.ACE inhibitory activity was measured with 5 mM HHL as a substrate by spectrophotometric method according to Jakubczyk et al. . Briefly50 (inhibitory concentration) was determined by assessing the ACE inhibition of several dilutions of each hydrolysate sample and interpolating the peptides concentration at which the inhibition percentage reached 50%. The IC50 value was calculated from the graph plotting inhibition for the five different peptide concentrations. Enalapril, applied in hypertension and chronic failure treatment as inhibitory of ACE, was used as the positive control.The ICThe sample obtained after absorption process was separated using ion exchange chromatography on DEAE-cellulose (the column 1.5 × 14 cm). The concentration of peptides for ion exchange chromatography was calculated by soluble peptide content by the trinitrobenzenesulfonic acid (TNBS) method using L-leucine as the standard . 2 mL ofPeptide fraction with the highest activity of ACEinhibiting properties was subsequently separated on Sephadex G10. 2 mL of fraction was loaded on the column (1 × 120 cm) previously washed with distilled water that was used also as an eluent, with flow rate of 0.8 mL/min. One milliliter of each fraction was collected and the absorbance was monitored at 220 nm. Fractions with the highest absorbance were combined and ACE inhibitory activity was determined. Fractions with the highest ACE inhibitory activity were lyophilized and used in further analysis.Km), the inhibition constant (Ki), and the maximum velocity (Vmax) of ACE. The kinetic parameters were evaluated by Lineweaver-Burk's method. The reaction conditions were the same as ACE inhibitory activity assay. Briefly, 50 μL of peptide sample (or 0.1 M borate buffer pH 8.3 for enzyme activity) was added to 50 μL HHL solution  and 50 μL of 3 mU/mL ACE (one unit of ACE activity was defined as an increase absorbance of 0.001 per minute at 390 nm). The reaction mixture was incubated at 37°C for 60 min. The reaction was terminated by adding 0.7 mL of the 0.1 M borate buffer with 0.2 M NaOH. The absorbance was measured at 390 nm.Hippuryl-His-Leu solutions  were prepared and used to determine the Michaelis constant (μg) were hydrolyzed in gas phase using 6 M HCl at 115°C for 24 h. The liberated amino acids were converted into phenylthiocarbamyl (PTC) derivatives and analyzed by high-pressure liquid chromatography (HPLC) on a PicoTag (3.9 × 150 mm) column  according to method of Boogers et al. [The samples , 50–2500 m/z (product ion), and collision energy (CE): 51.2 V. Acquired raw data were processed by Mascot Distiller followed by Mascot Search  against NCBInr database (20120224). Search parameters for precursor and product ions mass tolerance were 40 ppm and 0.6 Da, respectively, enzyme specificity was semitrypsin, missed cleavage sites allowed were 0, and fixed modification of cysteine was by carbamidomethylation and variable modification of lysine carboxymethylation and methionine oxidation. Peptides with Mascot Score exceeding the threshold value corresponding to <5% false positive rate, calculated by Mascot procedure, and with the Mascot score above 30 were considered to be positively identified.The molecular mass andpeptide sequencing were estimated by positive ion mode using electrospray ionisation-mass spectrometry. Lyophilized samples (0.1 mg) were dissolved in 0.3 mL of 50% acetonitrile containing 0.1% formic acid (v/v) and analysed using Applied Biosystems mass spectrometer, type Star XL MS-MS with nanoelectrospray ionization (nano-ESI). The analysis was carried out in duplicate in the positive ion mode under the following conditions: the potential of the cone (DP): 60 V, the voltage on the needle during sputtering (IS): 900–1500 V, and scan mode: TOF-MS (product ion). Ions were monitored in the field: 100–3500Each treatment was conducted in triplicate and the results were presented as mean ± standard deviation. STATISTICA 7.0 was used for statistical analysis. Tukey's test was used to estimate significant differences among the mean values at the 5% probability level (α < 0.05).Most studies of bioavailability of peptides released during protein hydrolysis process relate to changes caused by a single proteolytic enzyme or subsequent hydrolysis resulted in proteases of the digestive system, bypassing the step α-amylase hydrolysis. The α-amylase is not a proteolytic enzyme, but, in order to reflect the processes occurring in the gastrointestinal tract more accurately, it was also used in this study. The globulins hydrolysates obtained from pea were digested under simulated gastrointestinal digestion condition . The degree of hydrolysis was noted as follows: after pepsin, 14.42 ± 0.14%, and after pancreatin, 30.65 ± 0.33%. Similar results were obtained by Lo et al.  for soyb in vitro digestion and absorption, the IC50 ACE peptides inhibitory value was noted to be 0.72 mg/mL, as enalapril with IC50 value of 7.46 mg/mL was used as a control. This result corresponds well with results obtained by Barbana and Boye [50 values were determined for two varieties of chickpea and yellow pea for nonhydrolyzed proteins and after gastrointestinal digest. The results showed that only hydrolysates have ACE inhibitory activity and IC50 were noted for chickpeas to be 0.229 mg/mL and 0.14 mg/mL and for yellow pea 0.159 mg/mL [50 = 0.77 mg/mL and 0.26 mg/mL, respectively. As it was previously indicated, the legumes food proteins are a good source of ACE inhibitory peptides released after digestion and they must enter the circulatory system to have benefit to health.Potentially bioactive fragments of proteins with structural motifs corresponding to the bioactive peptides are inactive precursor protein in the sequence and only released by the action of proteolytic enzymes which may exhibit diverse biological activity , 25. In and Boye , where I59 mg/mL . Akillio59 mg/mL  reported50 value was noted to be 0.0014 mg/mL. It should be noted that in the peptide content in most peptide fractions no significant difference was determined. F8 was not characterized by the highest peptides content; it suggests that the ACE inhibitory activity depends on the amino acids sequences. This fraction was separated using gel filtration on Sephadex G10 , DLKLP (585.56 m/z), GSSDNR (634.56 m/z), MRDLK (662.59 m/z), and HNTPSR (711.59 m/z). An important step in the identification of peptides with high ACE inhibitory properties is determination of their amino acid composition. In this study, amino acid composition of peptide fractions with the highest ACE inhibition activity obtained after the separation on Sephadex G10 was determined by hydrolyzing them using 6 M HCl and then analyzed by HPLC . Due to Ki of the peptide fraction resulted in reduction of the value of Km and the maximum rate of enzymatic reaction (Vmax); therefore, this fraction was uncompetitive inhibitor of ACE. This means that inhibitor binds only to the enzyme-substrate complex (E-S) and not to the free enzyme. Although ACE inhibitory peptides have been reported as competitive inhibitors [Lineweaver-Burk plots of the ACE inhibition pattern of the fraction B were shown in hibitors , 25, moshibitors , two ACEhibitors , 32.Km, Vmax, and Ki and mode of inhibition were determined. This fraction belongs to uncompetitive inhibitor of ACE activity.As a result of the conducted research, it can be stated that the seeds of pea are the source of precursor protein, which releases the ACE inhibitory peptides as a result of enzymatic hydrolysis. In this study, ACE inhibitory peptides were identified by ESI-MS/MS. The sequences of novel ACE inhibitory peptides were GGSGNY, DLKLP, GSSDNR, MRDLK, and HNTPSR. Based on Lineweaver-Burk plots for the fraction B, the kinetic parameters as


Chronic myeloid leukaemia (CML) is a myeloproliferative disorder promoted by the constitutive tyrosine kinase activity of Bcr-Abl oncoprotein. Although treatment with the Bcr-Abl-inhibitor imatinib represents the first-line therapy against CML, almost 20-30% of patients develop chemotherapeutic resistance and require alternative therapy. Here we show that a strong hyper-phosphorylation/activation of ERK1/2, Akt Ser473, and 40S ribosomal protein S6 (rpS6) is detectable in imatinib-resistant KCL22 and K562 CML cells as compared to the -sensitive cell variants. In imatinib-resistant CML cells, high concentration of imatinib is required to strongly inhibit Bcr-Abl, ERK1/2 and Akt Ser473 phosphorylation, but under these conditions the phosphorylation of rpS6, a common downstream effector of MEK/ERK1/2 and PI3K/Akt/mTOR pathways is only slightly reduced. By contrast, down-regulation of the protein kinase CK2 by the inhibitor CX-5011 or by silencing the CK2 subunits does not affect the activation state of MEK/ERK1/2 or PI3K/Akt/mTOR signalling, but causes a drop in rpS6 phosphorylation in parallel with reduced protein synthesis. CK2-inhibition by CX-5011 induces cell death by apoptosis and acts synergistically with imatinib or the MEK-inhibitor U0126 in reducing the viability of imatinib-resistant CML cells. The ternary mixture containing CX-5011, imatinib and U0126 represents the most effective synergistic combination to counteract CML cell viability.These results disclose a novel CK2-mediated mechanism of acquired imatinib-resistance resulting in hyper-phosphorylation of rpS6. We suggest that co-targeting CK2 and MEK protein kinases is a promising strategy to restore responsiveness of resistant CML cells to imatinib. BCR-ABL1 fusion oncogene , which gives rise to the 1;q34], w2q11;q34)oncogene . Althougoncogene , 6, 7. Toncogene , 8, 9.Protein kinase CK2 is a highly conserved and constitutively active Ser/Thr protein kinase, which is usually present as a tetramer composed of two catalytic (α and/or α’) and two regulatory (β) subunits. This protein kinase is distributed in all subcellular compartments, where it phosphorylates a huge number of protein substrates implicated in fundamental cellular processes –12. CK2 This study provides new insights into molecular mechanisms of imatinib-resistance related to CK2 in CML KCL22 and K562 cell lines, where the drug treatment does not induce an up-regulation of the kinase. Particular attention is focused on MEK/ERK1/2 and PI3K/Akt/mTOR survival pathways to highlight a potential CK2-mediated hyper-activation induced by imatinib-resistance. The potent and very selective CK2-inhibitor CX-5011 is used in combination with imatinib and the inhibitor of MEK to define new therapeutic strategies able to overcome imatinib-resistance.BCR-ABL1 gene amplification or by mutations of the Bcr-Abl kinase domain ATP by recombinant CK2 holoenzyme. The outcome that rpS6 is not phosphorylated by CK2 (data not shown) suggests that the kinase does not act directly on the ribosomal protein.To further analyse the mechanism underlying the regulation of rpS6 phosphorylation by CK2, we examined whether the phosphorylation of the canonical rpS6 phospho-sites might be influenced by a potential CK2-catalyzed phosphorylation of the ribosomal protein at different site(s). To this purpose the protein was immunoprecipitated from R-KCL22 cell lysates, dephosphorylated by lambda phosphatase and subsequently phosphorylated Since rpS6 phosphorylation is essential for protein synthesis and cell growth , we veriIt has been reported that the inhibitors of the MEK/ERK1/2 pathway, including U0126, can be classified as cytostatic but not as cytotoxic anticancer drugs  and thatTo further analyse the mechanism of cell death triggered by CX-5011, we evaluated the nucleosome formation in both imatinib-sensitive and -resistant KCL22 cells. Figure We have recently found that co-treatment with imatinib plus CX-4945, a CK2-inhibitor of the same class of CX-5011 but less selective, causes a synergistic effect in reducing the cell viability of imatinib-resistant CML cell lines . It has We have previously demonstrated that the protein kinase CK2 is over-expressed in imatinib-resistant LAMA84 cells as compared to the -sensitive variant and that CK2 up-regulation strengthens the imatinib-resistance phenotype conferring survival advantage against imatinib . Our resThe specific role played by Bcr-Abl or CK2 on the pathways up-regulated upon imatinib-resistance was evaluated treating R-KCL22 cells with imatinib or CX-5011, respectively. As expected, higher concentration of imatinib is required in imatinib-resistant CML cells to counteract the constitutive activation of Bcr-Abl in parallel with the hyper-phosphorylation of ERK1/2 and Akt Ser473, while the same concentration is quite ineffective on the phosphorylation extent of rpS6 Figure . Previouvs cytoplasmic localization of p70S6K . Another hypothesis is that imatinib-resistance might trigger an alternative p70S6K-independent and CK2-mediated pathway leading to rpS6 phosphorylation. On this point, evidence has been provided for an additional rpS6-phosphorylating kinase not identified so far -L-methionine/cysteine protein labelling mix was from Hartmann Analytic . Protease inhibitor cocktail was from Calbiochem , while phosphatase inhibitor cocktails from Sigma-Aldrich . Imatinib mesylate was from Cayman Chemical , CX-5011 from Glixx Laboratories , rapamycin and U0126 from Selleck Chemicals  and PF-4708671 from Sigma-Aldrich. RRRADDSDDDDD peptide was kindly provided by Dr. Oriano Marin . Anti-CK2α ATP (about 1000 cpm/pmol). Assays were stopped by absorption onto phosphocellulose filters. Filters were washed four times in 75 mM phosphoric acid -L-methionine/cysteine protein labelling mix . After 1 h radiolabelled medium was removed and cells were washed and lysed as above described. Exposure to CX-5011 was maintained throughout the experiment. Similar amounts of radioactive lysates were subjected to SDS-PAGE and blotted. The membrane total labelling was measured using the Cyclone Plus Storage PhosphorSystem.5 × 106), pre-treated with DMSO or 3μM CX-5011 for 4 h, were washed twice with PBS. Cells were then resuspended in 300 μl of ice-cold hypotonic buffer containing 20 mM Tris-HCl, pH 7.8, 1% (v/v) Nonidet P-40, and protease and phosphatase inhibitor cocktails. After 5 min, samples were diluted 1:1 with water and the solution passed through a 23 G needle 10 times using a 2 ml syringe. Samples were immediately centrifuged at 100 × g for 10 min at 4°C. Cytoplasmic fractions were collected as supernatants, while the pellets containing the nuclear fractions were extracted with 100 μl of lysis buffer A.R-KCL22 cells  was added to each well. Incubations were stopped by addition of 20 μl of a pH 4.7 solution containing 20% (w:v) SDS, 50% (v:v) N,N-dimethylformamide, 2 % (v:v) acetic acid and 25 mM HCl. Plates were read at λ 540 nm absorbance, in a Titertek Multiskan Plus plate reader . DC50 (concentration inducing 50% of cell death) values were calculated with Prism 4.0c software.Cell viability was detected by the method of MTT [3--3,5-diphenyltetrazolium bromide), incubating 4 × 10Effect of combined inhibitors was assessed by treating cells with increasing concentrations of drugs, added at fixed ratio, as indicated. The combination index (CI)  for the t-test. Values of *p < 0.05 or **p < 0.01 were defined as statistically significant.Data are presented as means ± SD and mean differences were analysed using


Endothelial cells inhibit blood platelets by releasing nitric oxide (NO) and prostacyclin (PGI2). NO and PGI2 activate cGMP and cAMP synthesis, leading to the activation of cGMP- and cAMP-dependent protein kinases , phosphorylation of substrate proteins, and inhibition and reversal of platelet activation and aggregation. A known target of PKA and PKG is the small G-protein Rac1. The active form of Rac1, Rac1-GTP, contributes to cytoskeletal reorganization, platelet spreading, adhesion, granule release, and aggregation. PKG and PKA activation have been shown to reduce Rac1-GTP levels. Rac1-GTP is controlled by GTPase-activating proteins (GAPs - reduce Rac1-GTP) and Guanine-nucleotide exchange factors (GEFs - elevate Rac1-GTP). We hypothesized that specific GAPs and/or GEFs might mediate the inhibitory effects of PKG and PKA on Rac1. Screening for potential substrates among the GAPs and GEFs expressed in platelets resulted in the identification of the GAP ARHGAP17  as well as the GEF ARHGEF6  as new substrates of PKG and PKA in human platelets. We mapped the phosphorylation sites on ARHGAP17 and ARHGEF6, and we investigated the functional consequences of phosphorylation. Phosphorylation of ARHGAP17 by PKA resulted in a dissociation of ARHGAP17 and its interaction partner CIP4. Phosphorylation of ARHGEF6 by both kinases resulted in the association of the ARHGEF6/GIT1 complex with the phospho-serine/threonine binding protein 14-3-3.Our data suggest that PKG- and PKA-mediated phosphorylations of ARHGAP17 and ARHGEF6 lead to a reorganization of protein complexes involved in the control of Rac1. These events are likely to contribute to Rac1 inhibition by cyclic nucleotides. Similar findings regarding the regulation of a critical small G-protein by GAP and GEF phosphorylation have been described for Rap1 in platelets before. We conclude that phosphorylation of GAPs and GEFs of small G-proteins might represent a general principle of platelet regulation by cGMP and cAMP.


Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.  High-throughput selection methods, such as fluorescence-activated cell sorting or automated colony pickers, normally use some expensive procedures based on fluorescence. Vector engineering and site-specific recombination have shown acceptable results in isolating high-producing clones[2]. The improvement of impressive and safe non-viral vectors would considerably facilitate the complexity of recombinant protein expression. Site-specific recombination integrates transferred gene into a site with the desired surrounding chromatin. There are several commercially available systems for site-specific integration as follows: 1) two groups of recombinase family that include the tyrosine recombinase and serine recombinase[3]. Both families direct recombination between two recombinase target sites and result in sequence-specific DNA insertions. Since 1990s, tyrosine recombinases, such as Cre and Flp, have been used for site-specific integration in animal cells[4]. According to the reports, these particular recombinases can cause chromosomal aberrations. Because of the presence of pseudo-sites, control of inaccurate effects is less powerful[5]. Different attempts have been made to develop the efficiency and specificity of this system by redesigning their site specificity. 2) Class II transposable elements, including the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons that move in the host genome via a “cut and paste” mechanism. They account the most useful tools for genetic engineering because of their easy laboratory handling and controllable nature.The isolation of high-producing Chinese hamster ovary (CHO) clones for biopharmaceutical products is an industrial problem. Suitable stable cell lines are widely generated for high-level expression of recombinant proteins using random integration linking genomic amplification; however, it is very laborious and time-consuming and needs improvement. Also, due to position effects, it results in low and unpredictable yields of expression[8]. Use of a site-specific recombinase may be expensive, and initial screening for amplifiable sites is necessary to be performed. This step is performed only once to produce an isogenic cell line that can be utilized to produce a range of desired gene products. The reviewed methods have also obvious positive effects on cell-specific productivity.These strategies can increase biopharmaceutical protein titer and decrease the required time to achieve sufficient amounts of protein for pre-clinical evaluationTransposases[9].DNA transposons are natural genetic elements moving via a conservative cut and paste mechanism from one chromosomal location to another. They are composed of an open reading frame coding for the transposase and flanked by two inverted terminal repeats. The molecules that support the integrative process are relatively easy for engineers since only two separate plasmids are co-delivered to the cells . The don[10]. SB target site selection is determined by structural constraints rather than primary DNA sequence. In contrast to most retroviral-based vectors, SB integrates somewhat randomly in mammalian cells, without any discernible preference for actively transcribed genes[11]. This feature makes SB a suitable candidate for development into targeted systems. The first step towards specific site integration at the genome scale was achieved in 2007 with an SB transposase fused with the E2C zinc-finger protein. However, these systems are still based on random integrations[12]. SB is a non-viral element that can integrate efficiently into the mammalian host chromosomes. The transposition of SB element always occurs into a TA target dinucleotide, which is subsequently duplicated upon insertion by cellular DNA repair pathways[13] and less preventive effect on transposition. Tol2 transposase activity is more susceptible to be affected by molecular engineering, as described for SB, and its targeting preference is not obvious. Also, 5’ regions of genes are most preferred for Tol2, the same as other hAT elements[14].Tol2, a hAT superfamily, is another transposon system that can transfer large transgenes of up to 11 kbp with minimal loss of transposition activity[10]. It then excises the entire transposon from its original location and catalyzes its integration at another chromosomal site through a mechanism that is not dependent on host factors[11]. In comparison, PB is an alternative to SB and even to Tol2 because of its large cargo size (up to 14 kb), high activity in many cell types, long-term expression in mammalian cells, less susceptibility to be affected by molecular engineering, and the ability to excise precisely[15]. PB, a superfamily of PB, is a DNA transposon isolated from the cabbage looper moth. It integrates into TTAA sequences, and transposition occurs via a cut and paste mechanism in which the PB transposases are initially recognized and bind to the transposon termini[16]. It seems that host factors have an important role in PB and Tol2 integration rates. Also, the potential risk of undesirable integration cannot be excluded and the continuous expression of an inadvertent integrated transgene can result in genotoxic risk[17]. Transposable element, such as PB and SB, have been used for integrating recombinant genes into cultivated mammalian cells[18-20]. Using PB transposon, recombinant CHO cell lines are able to produce up to four-fold more recombinant protein in comparison to standard transfection and are stable up to three months in the absence of selection[21]. PB and Tol2 have shown similar error rates in insertion into genes, especially introns or getting close to transcription start sites. There are some pieces of evidence that active chromosomal regions favor the PB and Tol2 integration processesCre/loxP recombination [22]. Cre recombinase recognition site, known as a loxP site, is a 34-bp DNA sequence that will remain after integration. Therefore, this reaction is reversible by the loxP site. Despite this disadvantage, the Cre/loxP system is able to generate high specificity by about 80%[23]. In an experiment, dhfr-deficient CHO cells were transfected with a vector containing the green fluorescent protein reporter and a dhfr gene downstream of loxP site[22]. The selection of positive clones was first based on the highest fluorescence and in the second step, methotrexate amplification-selection procedure was performed. Then, a targeting vector containing the antibody of light and heavy chain genes was fused to a hygromycin resistant marker and a loxP site was co-transfected along with Cre recombinase to catalyze the site-specific recombination. The targeting vector was integrated into a locus that was transcriptionally active and amplifiable  and an FRT sequence for subsequent recombination into the transcriptionally active sites, cells were screened for different gene integration sites. Then 20 candidate clones were selected for amplifiable expression sites, three of which were suitable as hosts for Flp recombination of antibody genes. After six days of culturing, up to 200 mg/L full-length anti-CD20 antibody was produced in a spinner flask. Other report have shown that this method can be used in a broad range of target genes. In other similar works utilizing the Flp/FRT system[24], a target vector was tagged by FRT and an antibiotic resistance marker. Also, high producing clones were isolated for tissue plasminogen activator, and the highest specific productivity achieved was 17.1 μg/106 cells/day. Therefore, this method is very similar to the Cre/lox system with the same advantages and limitations. Another gene-targeting system is known as Flp/FRT by Flp recombinase and gene sequences tag with a FRT sequence. Flp/FRT system has weak recombination specificity (10%)C31 integrase recombinationΦattP site and the bacterial host attB site. There are up to 100 target sites for this system in the human genome[27]; however, the selection of specific inegration sites in this system is impossible. The high rate of chromosomal translocations and the risk of gene-toxic effects such as cancer are important concerns related to ΦC31. The obvious advantage of ΦC31 integrase over Cre and FRT systems is its irreversibility. The ΦC31 integrase catalyzes recombination between attP and attB sites, which consist of different sequences. Two resulting recombination hybrid sites cannot be substrates for the integrase activity [28]. There are many integrases that are functional in mammalian cells and have been commonly used in combinatorial systems, such as Bxb1, R4 Cre, and Flp . Artificial ACE is composed of a mammalian based artificial chromosome (Platform ACE), an ACE-targeting vector, and a mutant λ integrase (ACE integrase) for site-specific recombination. The platform ACE consists of tandem repeated ribosomal genes, repetitive satellite sequences (from the pericentromeric heterochromatin), natural centromeres, and telomeres to enable DNA replication without need for integration into the host cell genome, which reduces clonal heterogeneity because of chromosomal aberration. Recombination acceptor of attP sites in platform ACE is about 50–70, thus allowing for the incorporation of multiple copies of the considered gene. The platform ACE cell line is cotransfected with the ACE-targeting vector and ACE integrase plasmid. Following the recombination event, promoterless selection marker on the ACE-targeting vector integrates the downstream of the SV40 promoter in the platform ACE. Therefore, under selection pressure, the survived cells are identified as clones that have undergone a correct recombination event /Cas system, which uses re-programmable trans-activating CRISPR-RNA for sequence-specific cleavage, has emerged as an efficient tool for genomic modifications[62-65], numerous studies are nowadays dedicated to establishing efficient, targeted gene integration systems. There are different enzymes that have to be engineered to induce and target a double-strand break for recombination. Mammalian cell protein expression system has been the dominant recombinant protein production system for clinical applications in last two decades. This system has produced more than half of the biopharmaceutical products in the market and several hundreds of candidates in clinical development. Furthermore, many improvements have been made in cell line engineering methods, genetic methods of expression, gene silencing, and gene-targeting systems. Due to inefficiencies of traditional random integration methods in producing appropriate yields expression in industrial scalesNew site-specific non-viral vectors based on a given recombinase, integrase, or modified transposase can direct integration into a related site with a similar nucleotide sequence and thus correct random integration limitations and immunogenicity in gene therapy vectors.Tol2, increase integrated gene copy numbers and improve the recombinant protein titer as compared to the standard transfections[66]. However, the random inherent of transposases is a major drawback for their genomic modification applications. Phage recom-binases  enable an efficient and a site-specific integration of transgene and are more easily vectorizable. The in vitro GATEWAYTM cloning method (invented by InvitrogenTM USA) with an impact on a variety of research areas benefits from phage integrases[67]. The site-specific non-viral vectors based on the ΦC31 integrase have been promising in achieving long-term therapeutic gene expression through integration in tissue culture cells and in animals. However, these systems are limited by the need for a prior establishment of platform cell lines and the possibility of chromosomal aberrations[68]. The artificial engineered mammalian chromosomes are an example for an ideal gene delivery vector, with a stable episomal maintenance and a large transgene capacity. However, some factors, such as having a fragile construct, difficulties for purification, and low transduction efficacy, limit the application of human artificial chromosomes[69]. There is still much need for efforts to further improve the methods for designing recombinases with altered specificity and with combinatorial structures that would be more practical. Therapeutic potential of targeted recombination systems and optimal methods for delivering these enzymes into different cell types are the other fields that need continued exploration. Transposon-based vectors, such as SB, PB, and [70] also represent challenges for this system. The latest improvement of gene-targeting methods for mammalian recombinant proteins expression, as an important subcategory of cell line development, was summarized in this review. In addition to above mentioned methods, the recent tool CRISPR/Cas could be an advantageous choice for the future, but features such as off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages


Increased regulatory T cell (Treg) numbers within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Also, Treg increase in cancer patients have been associated with reduced survival and inhibition of anti-tumor immune responses. Therefore, decreasing the numbers and/or function of Tregs is needed to facilitate better outcomes for cancer patients. The phosphoinositide 3-kinase (PI3K-Akt) pathway plays important roles in cell growth, survival, and proliferation of T cells. However, little is known about the role of different isoforms of PI3K in different T subsets activation. Here, we explore the role of PI3K isoforms in Tregs and conventional T cell activation with the intention of identifying potential differences to selectively inhibit Tregs.in vitro analysis, FACS-sorted Tregs and Tconvs from Foxp3-GFP mice were stimulated (anti-CD3 Ab/anti-CD28 Ab/IL-2) with or without inhibitors for 72 hrs. Phosphorylation of Akt (S473) and S6 was analyzed by flow. Violet Cell Trace (VCT) proliferation assay was performed using flow cytometry. The in vivo effect of inhibitors on frequency of different T cell subsets were assessed in TC-1 tumor-bearing mice on days 3 and 6 after treatment.For We found that pharmacologic inhibition of PI3Kδ isoform but not PI3Kα or PI3Kβ exhibit a selective blockade of PI3K/Akt signaling and the inhibition of proliferation of Tregs. In contrast, the inhibition of each individual Class IA PI3K isoform in conventional T cells did not affect their activation and proliferation. Further, our data on combined inhibition of PI3K isoforms suggest that these isoforms are redundant and compensate for each other in Tconvs. Interestingly, PI3Kα or PI3Kβ were unable to compensate for PI3Kδ in Tregs, making PI3Kδ inhibition a selective Treg-targeting approach. Importantly, we also show that inhibition of PI3Kδ and not PI3Kα PI3Kb in tumor-bearing mice significantly decreases the level of Tregs without affecting other T cell subsets. We believe that our findings provide a basis for development of novel cancer immunotherapies based on selective inhibition of Tregs.


In recent decades, with the convergence of high-resolution anatomical and physiological techniques, a perspective is emerging on inhibition in the nervous system that recognizes the vast diversity of functions it serves. These include roles in modulation, development, and plasticity, in addition to the common perception of inhibition as spike suppression. Progress toward this more nuanced understanding of inhibition has derived from many studies across the nervous system, but here we focus on part of the brainstem auditory system, where discrete inhibitory nuclei interact in unique and fascinating ways to integrate and compute binaural information in the circuitry for sound source localization.We have solicited studies for this special topic on inhibition in the auditory brainstem circuitry from laboratories around the world. The assembled manuscripts provide an authoritative collection of concepts across the breadth of neuroscience research on inhibitory function that focus on three major themes.A major advantage of investigating inhibition in the auditory pathway is the distribution of inhibitory centers among its subthalamic nuclei. In general, these nuclei are involved in computing the azimuth location of a sound source, by integrating the acoustic stimulus from both ears. The superior olivary complex (SOC) is the first region of the brain to compute sound location by comparing the input to the two ears. The MNTB is central to this circuitry and is highly specialized; being driven by the Calyx of Held (one of the largest synapses in vertebrates) and providing a powerful glycinergic inhibition to its targets. Despite its pivotal role in this circuit and extensive investigation, our understanding remains incomplete, the details of its inputs, output, and sound encoding are still being explored.in vitro study, Roberts et al. . Surprisingly, development of immunohistochemical markers for glycinergic transmission, although delayed, reach typical levels in adulthood, demonstrating remarkable developmental plasticity in this system and provide evidence for alternative sources of inhibitory input.The MNTB appears to be so central to auditory circuitry, that it is difficult to imagine how the network could adapt to its absence, but genetic tools have allowed Altieri et al.  to addreSynaptic plasticity allows developmental change and activity-dependent adaptation of information transmission throughout the nervous system. In the auditory pathway, myriad examples of plasticity of inhibitory signaling are demonstrated. They include classical forms such as LTP, LTD, depression and facilitation, as well as novel forms described below.Plasticity is a prominent feature of processing in another region of the auditory brainstem, the dorsal cochlear nucleus (DCN), which includes complex intra-CN inhibitory circuitry driven by the auditory nerve via the tuberculoventral interneuron. Sedlacek and Brenowitz  carefullB receptor-dependent long term depression, especially early in development. In their current paper, they add to this body of work by demonstrating that this synapse also expresses long-term potentiation, but somewhat later in development. This potentiation surprisingly also depends on GABAB receptor function.Long-term synaptic plasticity is a hallmark of excitatory synaptic coupling, particularly during development. In contrast, long-term plasticity at inhibitory synapses is less commonly studied. One exception is at the MNTB-LSO synapse where Kotak and Sanes  have pre− reversal potential. The NO-dependent depolarizing shift in reversal potential demonstrates a possible means to modulate inhibition in SPN neurons, without influencing inhibition in other collateral targets of the MNTB.Plasticity of inhibition can also occur indirectly, as demonstrated in the SPN, a synaptic target of the MNTB. Yassin et al.  in a commature auditory circuitry may be more complex than previously appreciated. In this issue, Case et al. (Inhibitory neurons of the SOC typically release two or more transmitters in early development, but revert to a single dominant transmitter following hearing onset. This general principle has been refined in the last two decades. Recently however, it has become apparent that transmitter release in e et al.  extends e et al.  and Nerle et al.  extend re et al.  use opti− and K+ conductances occurring in both pre and postsynaptic compartments of this circuitry.One underappreciated aspect of inhibition that is emerging in the literature is that classical synaptic inhibition can engage voltage gated ion channels and signaling pathways beyond their classical receptors. Hamlet et al.  investigFinally, this Research Topic presents two review offerings. The first, from Ohmori  focuses Together these studies and perspectives provide a taste of current concepts, with promises of more exciting insights into auditory function around the corner. Inhibition is nonetheless important for its suppression of mere excitation, and as we see here this field is vibrant and forward-looking. We hope that neuroscientists investigating the physiology of inhibition beyond the auditory system will find this work equally exciting and we thank all of our contributing authors for their excellent work. And to you, our readers, we hope you find some inspiration for your own research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.