Degranulation of mast cells and basophils, with release of agents of the allergic response, ensues when multivalent antigens bind to and cross-link the cells’ receptor-bound IgE antibodies. A widely used commercial monoclonal IgE antibody, SPE-7 IgE from Sigma, was found to possess the radically anomalous property, termed “cytokinergic”, of inducing basophil degranulation without the intervention of an antigen. We show here that the IgE monomer, freed of protein contaminants, is devoid of this activity, and that the source of the anomaly is a trace impurity, identified as a dissociation-resistant IgE trimer. Possible models for the formation of IgE trimers and the manner in which they cross-link cell surface receptors are suggested herein. These antibodies bind tightly to high-affinity receptors (FcεRI) on mast cells in surface tissues, or on basophils in the blood, to sequester the parasite antigens or toxins and evoke an immediate and potent immune response. A central feature of this mechanism is cross-linking of the receptor by attachment of a multivalent antigen to two or more receptor-bound IgEs, thereby delivering the signal for mast cell or basophil activation3. The cost of the evolution of this protective mechanism in mammals is the risk of sensitization to otherwise harmless antigens  with the development of allergic disease. IgE binding to the receptors in the absence of antigen should not induce cell activation, which would serve no purpose in adaptive immunity, and could indeed pose a severe danger. The discovery of several monoclonal IgEs that stimulate mast cell and basophil activation without the need for an antigen was therefore perplexing4.The function of IgE antibodies in adaptive immunity is to erect an effective barrier against infection by multi-cellular parasites and the toxicity of animal venoms7. Kitaura et al. further established that some monoclonal IgEs could induce cell activation in the absence of antigen. Different monoclonal IgEs could be classified according to their antigen-independent activities, for which they coined the term, “cytokinergic activity”4. The higher this cytokinergic activity of the IgE, the greater the variety and potency of the activities it evoked. Monoclonal IgEs were grouped according to whether they were highly cytokinergic (HC), moderately (MC), or poorly (PC) so. Only IgEs in the first (HC) category could induce mast cell degranulation in the absence of allergen. The most highly cytokinergic IgE was the mouse NS1 hybridoma SPE-7 IgE, an anti-dinitrophenyl (DNP) antibody9. At 5 μg/ml the SPE-7 IgE alone activates mast cells and basophils to nearly the same extent as it does at 0.02 μg/ml in the presence of antigen, and by the same receptor cross-linking mechanism normally initiated by the antigen4.At the time of this discovery, it was already known that IgE at extremely high concentrations (5 μg/ml), some two orders of magnitude higher than that required for antigen-dependent cell activation, could up-regulate IgE receptor expression on mast cells10 to have concentrations of circulating IgE of up to 5 μg/ml and it was therefore suggested that antigen-independent (“cytokinergic”) IgE action may play a part in the pathophysiology of allergic diseases12. The inhibition of cytokinergic activity by free DNP, and more recently by recombinant SPE-7 IgE Fab, pointed to the Fv domain as the seat of the cytokinergic activity of the SPE-7 IgE antibody14.In some cases, sufferers from allergic disease were found by Bennich and JohanssonIn the present study, with the aid of higher resolution from a new size-exclusion column matrix, we show that SPE-7 IgE monomers are devoid of cytokinergic activity when isolated from all contaminants. This new technology has also enabled us to demonstrate the presence of a previously unrecognized component in SPE-7 IgE preparations: an SPE-7 IgE trimer that drives the cytokinergic activity. We thus resolve a long-standing issue concerning the nature of the cytokinergic phenomenon.To determine the origins of the exceptional cytokinergic action of SPE-7 IgE (Sigma), we isolated the pure monomeric IgE from the crude commercial product. For comparison we purified the equivalent monomeric IgE from the NS1 hybridoma (kindly provided by Dr. Zelig Eshhar) and recombinant SPE-7 produced in HEK cells using the sequence derived from the hybridoma. The recombinant SPE-7 was expressed both as wild-type mouse protein (mSPE-7) and as a mouse-human chimaeric form, comprising mouse SPE-7 IgE heavy- and light-chain variable regions and human constant regions , moderate (MC) or poor (PC)4.Reports that basophils or mast cells respond to high concentrations of certain monoclonal IgE antibodies date back to 198513. This was not observed in the earlier study with this antibody11, but was confirmed in subsequent published work28, and also here. The reason for this discrepancy has not been resolved.A single antibody preparation, the commercial mouse hybridoma SPE-7 anti-DNP IgE from Sigma, stands at the high-response extreme of the HC group, and causes not only pre-sensitization to antigen activation, but also degranulation of basophils and mast cells in the absence of antigenThis highly cytokinergic activity was confined to a small sub-group of the monoclonal IgEs, but it appears to violate a tenet of cellular immunology, that cell activation is initiated by cross-linking and clustering of, in this case, IgE-bearing surface receptors on binding of multivalent antigens. This raises the question of how such cross-linking might occur without intervention of a divalent or multivalent antigen.et al. and Kitaura et al.11, namely, the existence of a secondary (weak) IgE binding site in the Fab arms of the receptor-bound SPE-7 IgE. This would capture a free, unbound IgE from the supernatant that could attach itself to receptor-bound IgEs so as to form a cross-link, thereby simulating the action of a divalent antigen. In support of this scheme is the rapid termination of the HC activity by (1) removal of free SPE-7 IgE from the medium29, or (2) addition of monovalent DNP hapten or recombinant SPE-7 IgE Fab14. Furthermore, we have shown that the postulated cross-linking occurs through an Fv/Fv interaction14. These features are strongly reminiscent of an idiotype-anti-idiotype (Id-anti-Id) reaction. Intriguingly, SPE-7 IgE has been shown to display multi-specificity, binding not only to DNP, but also to a totally unrelated protein antigen31. The ‘conformationally labile’ CDRs may be responsible for the occurrence of this Fv/Fv (protein-protein) interaction.One plausible explanation was implicit in the results of Kalesnikoff 32. The authors envisaged an un-physiological situation in which a self-reactive monoclonal IgE, saturating the receptor binding sites on the cell surface, are exposed to a large excess of the same IgE. Self-association between the free and bound IgEs would be strongly enhanced by the large number (5 × 105) of the receptors and the mobility of the IgE-receptor complexes on the cell surface.A mechanism by which the exceptionally high expression of the FcεRI-bound monoclonal IgE might enhance even a low affinity interaction between the free and bound IgE was proposedAttempts in previous studies to free the SPE-7 IgE monomer of all traces of oligomers, which upon binding to the surface receptors could cause FcεRI clustering, were evidently insufficient to eliminate the trimers that we have now separated by affinity chromatography followed by a superior size-exclusion chromatography matrix Fig. .The primary outcome of the results presented here is that the IgE monomer in the widely used SPE-7 Sigma product has no detectable cytokinergic activity. It would not be surprising if the same conclusion were, by extension, equally true for other monoclonal IgE preparations falling into the HC class. Our results further suggest that the outcome of earlier work on supposedly cytokinergic IgE preparations may have been vitiated by inadequate resolving power of the methods available at the time and may need to be re-evaluated.33. However, in this recent study, as also in earlier EM investigations of self-associating (Id-anti-Id) or cross-linked  IgG37 or IgE molecules38, a preponderance of cyclic dimers, trimers or tetramers, rather than linear complexes, was observed. A feature of the IgG structure, missing in IgE, is the presence of a flexible “hinge” between the Fab and Fc regions; this allows the angle between the Fab arms to vary widely, permitting formation of a range of cyclic structures. However, the more restricted flexibility of IgE, lacking a hinge region, might be expected to limit the possible oligomers that can form. Cyclic oligomers are thermodynamically favoured provided that the structures are not strained in ring formation.The conclusion that a trimer is responsible for the degranulating action of the SPE-7 IgE preparation raised a number of new questions. We may ask in the first place why the trimer is the only IgE oligomer that is formed. A linear self-association process would lead to dimers, trimers and higher forms in predictable proportions, depending on the subunit concentration, as seen for example in negative-stain electron microscopy (EM) of antigen-IgE complexeset al.33, the relative disposition of epitopes presented on a model antigen dictated the types of complexes formed, including ring dimers or tetramers of IgE depending on the antigen. In the earlier Id-anti-Id study38, ring dimers and tetramers were again formed, although these consisted, respectively, of 1:1 and 2:2 complexes of IgE (Id-bearing) and IgG1 (anti-Id) antibodies. These studies indicate that IgE molecules alone can accommodate the formation of cyclic dimers or tetramers, and thus presumably also trimers, depending on the location and orientation of the epitope.A further key factor is the location of the epitope recognised by the self-associating antibody; in the study by Gieras 40 and found that they are more restricted in their conformations than the more flexible IgG Fab arms. Furthermore, the range of inter-Fab angles in the models agrees well with the measured EM images: 141° ± 34° 38. Thus we can infer that SPE-7 IgE trimer formation involves each antigen-binding site recognising a determinant that we have shown is located within the Fv region14; the precise location of the epitope and the restricted flexibility of the SPE-7 IgE Fab arms may confine self-association to trimers alone. The thermodynamic stability of a cyclic structure, as observed in the earlier EM studies, is apparently sufficient to survive dilution during chromatography.We have previously modelled the IgE Fab arms relative to the known structures of both free and receptor-bound IgE-FcWhile we have shown that the antigen-independent degranulating activity of SPE-7 resides in the trimer fraction, we cannot distinguish whether the trimers act directly upon the mast cells or interact with receptor-bound SPE-7 IgE monomers. This cannot be addressed experimentally since we have so far been unable to eliminate residual monomers from our trimer preparation. In any case, without knowing the precise structure of the trimer, it is impossible to predict whether the three SPE-7 IgE-Fc regions could engage three receptors simultaneously and lead to cell activation. In the unfractionated commercial SPE-7 samples, monomer predominates and must saturate the receptors, so that for the trimers to engage with receptor-bound monomer, we must assume that a cyclic trimer is capable of dissociation into an open structure.We are confronted next with the problem of the circumstances under which the trimers are formed. Local concentration must be assumed to play a part. The SPE-7 IgE is a mouse hybridoma, and is synthesized in mouse ascites cells or in tissue culture, where the concentrations may favour self-association of the IgE. There may be other features of the manipulations that the preparations undergo during synthesis, purification, concentration, freezing and storage.in vivo. Advocates of cytokinergic IgE have pointed out that the concentrations required for SPE-7 to provoke basophil degranulation may be reached in individuals infected by certain parasites or suffering from allergic disease. However, as previously pointed out32, HC IgE activity was discovered in experiments with monoclonal IgEs. Situations in which IgE antibodies of a single specificity are generated at high concentrations must be quite rare, perhaps confined to monoclonal responses in germinal centre-like structures in tissues. Nevertheless, it has been shown that certain monoclonal IgE antibodies, notably SPE-7, can elicit antigen-independent mast cell activation in the skin of mice during sensitization to chemical haptens41.A final question is whether IgE trimers exist The cell degranulating action of SPE-7 IgE observed here conforms to the original definition of cytokinergic IgE insofar as it is antigen (DNP)-independent, yet it seems to involve self-association in the manner of a trimeric Id-anti-Id complex, rather than being an activity of monomeric IgE molecules.8), and the immunoglobulin heavy and light chains for SPE-7 IgE were amplified from hybridoma cDNA by PCR as previously described14. The variable region sequences are deposited in GenBank (Accession nos. KJ734989 and KJ734990).RNA was extracted from SPE-7 IgE hybridoma cells .Polymerase incomplete primer extension (PIPE) cloning2. Following selection with 50 ng/ml hygromycin B (Invitrogen), cells were cultured in FreeStyle™ 293 expression media (Invitrogen), supplemented with hygromycin B, in spinner culture flasks. Culture supernatants were harvested and filtered through a 0.45 μm cellulose acetate filter.Human embryonic kidney cells  were transfected with purified plasmid DNA using PEI (Sigma-Aldrich) and cultured in Dulbecco’s Modified Eagle medium (DMEM), supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 U penicillin/streptomycin  at 37 °C and 5% CORecombinant murine and chimaeric SPE-7 IgE (rmSPE-7 and rchSPE-7) protein expression and DNP-specificity was confirmed using ELISA plates  coated overnight with 5 mg/ml DNP-HSA (Sigma-Aldrich), blocked with PBS-1% BSA, and incubated with cell-free culture supernatants for 2 hours. This was followed by addition of HRP-conjugated anti-human IgE detection antibody diluted 1/5000 (Sigma), or anti-mouse IgE detection antibody diluted 1/2000, and then avidin-HRP diluted 1/1000 (both R&D Systems). IgE was detected with TMB (R&D Systems) by measuring absorbance at 450 nm.8, were cultured in spinner culture flasks in Dulbecco’s Modified Eagle medium (DMEM), supplemented with 15% heat inactivated horse serum, 2 mmol/L L-glutamine, 10 U penicillin/streptomycin . Culture supernatants were harvested and filtered through a 0.45 μm cellulose acetate filter.SPE-7 IgE hybridoma cells  on an HPLC system (Gilson). For the designated experiments, the same purification methods (affinity chromatography and size exclusion) were used to purify the commercially available Sigma SPE-7 IgE and smaller contaminating proteins. Monomeric IgE antibodies were eluted at around 15 minutes and the contaminants were eluted after 20 minutes.The recombinant mSPE-7 and chSPE-7 IgE antibodies, and mSPE-7 IgE from the NS1 hybridoma culture were isolated from cell culture supernatants using an HiTrap NHS-activated HP column  amine coupled with IgG4-Fc(sFcεRIα)2 fusion protein, and the commercially available Sigma SPE-7 IgE.A new technology, the Superdex™ 200 Increase column (Amersham Pharmacia Biotech) was also utilized on the HPLC system (Gilson) to fractionate SPE-7 IgE produced from the NS1 hybridoma cell culture, which had first been isolated by means of the Fc(sFcεRIα)N-morpholino) propane sulfonic acid (MOPS) buffer  at 180 V for approximately 90 minutes. The gel was fixed and stained with Brilliant Blue G Colloidal stain (Sigma), and then de-stained to reduce background staining before a scanned image was recorded.SDS-PAGE electrophoresis was performed in 3- were performed on excised gel sections prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer coupled to a Proxeon EASY-nLC II nano-flow liquid chromatography system. Eluting peptides were ionised by electrospray ionisation. Precursor ions were surveyed between m/z 350–1800 at a resolution of 30000 and the twenty most intense ions were subjected to MS/MS by collision-induced fragmentation in the iontrap with a normalised collision energy value of 35.Raw data from the mass spectrometry analysis were processed using Proteome Discoverer™ software, version 1.4 with Mascot version 2.2 as the search algorithm. Initially, the data from gel bands 1–6 were searched against the uniprot_sprot_130220 database specifying mouse proteins (16597 entries). Then the data from selected gel bands, namely 1 and 4 only, was searched against an in-house custom database (128 entries), comprising sequences for SPE-7 IgE heavy chains and light chains derived from NS1-hybridoma cells. Finally, all database search results were reviewed within Scaffold 4.3.2 (Proteome Software) where protein identifications resulting from both the mouse protein database search and matches against the specific heavy and light chain SPE-7 IgE sequences, were filtered using a 95% confidence score and a minimum of three matched peptides to generate the overall protein sequence coverage.The three peaks isolated from the S200 Increase HPLC purification of NS1 mSPE-7 IgE were run again on the Superdex™ 200 Increase column (Amersham Pharmacia Biotech) to determine their molecular weight, using an inline miniDAWN light-scattering detector and an Optilab DSP Interferometric Refractometer, and the data was analysed using the ASTRA 4.9 software (Wyatt Technology).45), were cultured in RPMI supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 U of penicillin/streptomycin, and 50 μg/mL Geneticin  at 37 °C, 5% CO2. Cells were seeded at 1 × 104 cells/100 μl/well in a 96-well plate in complete media omitting Geneticin and cultured at 37 °C, 5% CO2 overnight before stimulation.RBL-SX38 cells, which express both the native rat FcεRI and the human tetrameric form of FcεRI  or following HPLC chromatography. Cross-linking of IgE-bound FcεRI was carried out by incubation with 0.1 μg/ml DNP-HSA diluted in HBSS/1% BSA (Sigma-Aldrich) for 1 hour at 37 °C, 5% CO2. Alternatively, mast cells were stimulated with up to 5 μg/ml recombinant murine C3a (Bio-Techne) alone or in combination with recombinant SPE-7 IgE.Following washing with HBSS/1% BSA, adherent cells were stimulated with IgE antibodies  for 4 hours at 37 °C, 5% CO2 and the lysates collected. β-hexosaminidase release was measured using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate (Sigma-Aldrich) as previously described46 and degranulation expressed as a percentage of the total for each well. Statistically significant difference between experimental conditions was determined by one-way ANOVA with Dunnett’s or Tukey’s post-test. GraphPad Prism  was used for all statistical analyses.After each incubation, supernatants were harvested for analysis. Cells were finally lysed with 100 μl/well HBSS/0.5% Triton X-100 (Sigma-Aldrich) for 30 minutes at 37 °C, 5% COAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary File


Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.Cryptotanshinone (CPT), a fat-soluble phenanthraquinone from one solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.Primarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.CPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK. The mTOR, a 289-KD serine/threonine protein kinase, plays a core role in multiple signaling pathways involving the mediation of cell growth, proliferation, apoptosis and autophagy, being considered a hotspot target for cancer therapy , 2. mTORActivation of the mTOR signaling is firstly initiated by a variety of cellular signals comprising mitogenic growth factors, hormones, nutrients, cellular energy, stress conditions and so on, then initiative signals are transmitted by many important node molecules via multiple pathways to regulate mTOR in final . A key pApart from the PI3K/AKT, AMPK, another important signaling pathway can also modulate mTORC1 . AMPK, aCryptotanshinone (CPT) is one of the four major fat-soluble tanshinones abstracted from Salvia miltiorrhiza Bunge widely used in traditional formula for treating a variety of diseases , 13. Up Here, we demonstrated by the molecular docking data that CPT unlike rapamycin has weak direct affinity with FKBP12, the structure domain of mTOR. Concurrently, CPT indicates no inhibition on PI3K and PTEN. Further results conclusively confirmed that activation of AMPK-TSC2 axis is dependent for CPT inhibition of mTORC1.Cryptotanshinone  was supplied by Xi’an Hao-Xuan Bio-Tech Co., Ltd. It was dissolved in anhydrous ethanol to form the stock solutions (20 mmol/L) stored at -20 °C. RPMI 1640 and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Mediatech . Fetal bovine serum (FBS) was from Hyclone , and 0.05% Trypsin-EDTA from Invitrogen . Type I insulin-like growth factor (IGF-1)  was rehydrated in 0.1 M acetic acid to prepare a stock solution (10 μg/ml) for equipartition and storage at -80 °C. Enhanced chemiluminescence solution was from Perkin Elmer Life Science . The antibodies used includes: 4E-BP1 , phospho-S6K1 (Thr389), S6K1, cyclin D1, Rb, AMPK, phospho-AMPKα(Thr172), ACC, phospho-ACC(Ser79), PDK1, phospho-PDK1(Ser241), PI3K(p110), PI3K(p85), phospho-PI3K(p85), PTEN, phospho-PTEN(Ser380), TSC1, TSC2, phospho-TSC2(Thr1462), , phospho-mTOR(Ser2448), mTOR, p-4E-BP1(T37/46), raptor, phospho-raptor(Ser792), rictor, mLST8 , AU1, HA , Flag, β-tubulin .+/+) and MEF (AMPK-/-) cells (mouse embryonic fibroblast cells) were generously provided by Dr. Shile Huang . Rh30, DU-145 and MCF-7 cells were cultured in antibiotic-free RPMI 1640 medium plus with 10% FBS, while MEF cells in antibiotic-free DMEM containing 10% FBS. All cells were maintained in a humid incubator . For serum-free experiments, monolayer cells were washed with fresh phosphate-buffered saline (PBS) and cultured in the serum-free DMEM.Human rhabdomyosarcoma (Rh30), human prostate carcinoma (DU-145), human breast cancer (MCF-7), MEF (AMPK4 cells/well (6 replicates) and grown overnight at 37 °C in a humidified incubator with 5% CO2. After CPT treatment for 48 h, 20 μL of one solution reagent was added into each well and the 96-well plate was continuously incubated for 4 h. Cell proliferation was determined by reading the optical density (OD) at 490 nm using a Wallac 1420 Multilabel Counter .Cells dispersed evenly in the medium were seeded in a 96-well plate at a density of 1 × 10http://www.rcsb.org/pdb/home/home.do). And downloading the 3D structure of rapamycin (CID:5284616), an inhibitor of mTOR and a positive control, and the docking molecule, cryptotanshinone (CID:496348) in the database of pubmed compound (http://www.ncbi.nlm.nih.gov/pccompound). Then the dehydration, hydrogenation and dehydrogenation for the protein and the molecules were executed using chimera software. In Autodock4.2 software, the semi-empirical potential function as the energy scoring function was used to score the small molecule conformation and location followed with Lamarck (LGA) genetic algorithm combined with local energy search. The size of receptor lattice box for docking is 2.25 nm × 2.25 nm × 2.25 nm(60 point × 60 point × 60 point), the lattice interval is 0.375 nm. The center of lattice box is located in the center of active site of mTOR protein. In the docking results, Similarity and free Binding energy for the dock site between cryptotanshinone and rapamycin were compared and analyzed.Searching and downloading the protein crystal structure of mTOR (2NPU) in the database of protein data bank (PDB) (Western blotting was performed as described previously . In brieThe immunoprecipitation assay was done as described previously . BrieflyRecombinant adenovirus encoding HA-tagged dominant negative AMPKα (Ad-dn-AMPKα) was a gift from Dr. Shile Huang . Initially, Rh30 cells were cultured in the growth medium, and the recombinant adenovirus was added for 24-h infection at 5 of multiplicity of infection (MOI = 5). Cells infected with Ad-GFP adenovirus alone served as a control. Expression of HA-tagged dn-AMPKα was confirmed by western blotting for HA band. The recombinant adenoviruses expressing FLAG-tagged wild-type mTOR, FLAG-tagged rapamycin-resistant (mTOR-rr) (mTORrr contains the entire mTOR gene with the site mutation (S2035I) that has intact mTOR kinase activity, but prevents rapamycin binding to FKBP12 and is rapamycin-insensitive , 17) andLentiviral shRNAs to AMPKα, TSC2 and GFP were described previously . For prot-test for multiple comparisons. The statistical significant difference was considered when p < 0.05.Quantitative data were expressed as Mean ± SE, and analyzed by one-way analysis of variance (ANOVA) with post-hoc Dunnett’s To understand whether CPT could directly binds to FKBP12, rapamycin binding domain of mTOR, Rh30 cells transfected with a rapamycin-resistant mutant mTOR (S2035I), mTORrr, that blocks FKBP12-rapamycin complex forming , were prPI3K, a bona fide upstream positive regulator of mTORC1, was routinely considered to mediate CPT inhibition of mTORC1 by deactivation. However, in the previous paper, we have found that CPT could up-regulate the expression of p-AKT (S473 & T308), leading to activation of AKT . Here, tThe major binding proteins of mTORC1 include mTOR, mLST8 and raptor . RapamycSince CPT has no affinity with FKBP12 and no effect on inhibiting the PTEN-PI3K pathway and disrupting the mTORC1, its inhibition of mTOR pathway then maybe due to intervention of upstream of mTOR, such as AMPK. So the expression of above protein kinase in cancer cells was measured using western blot assay. As shown in Fig. Compound C, inhibitor of AMPK, was added to further demonstrate CPT inhibiting mTORC1 was due to activation of AMPK. Based on Fig. To attain the further evidence on CPT activation of AMPK, Rh30 cells were infected with Ad-AMPKα-dn and lentiviral sh-AMPKα to downregulate the expression of AMPK. As expected, expression of phosphorylation of S6K1 was partially recovered in Rh30 cells expressing HA-tagged dominant negative AMPKα (Ad-AMPKα-dn), but not GFP (Ad-GFP), and the inhibition of 4E-BP1 was also reversed Fig. . Further50 from 2.45 μmol/L (AMPKα+/+) up to 16.14 μmol/L (AMPKα-/-)  Fig. . In addi/-) Fig. . This coTSC2 as a downstream of AMPK, was associated with TSC1 and formed a functional complex that inhibits mTOR . And theIt is well-known that mTOR has been extensively considered a very important target for cancer therapy, and many synthesized mTORC1 inhibitors, rapamycin derivatives such as RAD001, CCI-779, have been tested for cancer patients in clinic . FurtherRecently, many natural compounds from herbal drugs including cryptotanshinone , curcumiIn this study, it is firstly confirmed that CPT inhibits mTORC1 independent on binding to FKBP12, suggesting the effect of mTOR inhibition was due to signal transduction by regulation of its upstream Fig. . IGF-1 iThe main upstream of AKT-independent regulation of mTOR is AMPK/TSC2 pathway . AMPK beAMPK communicates the inhibitory signal to the mTORC1 by indirect and direct mode. TSC2 is phosphorylated and activated by AMPK mediating indirect inhibition on mTORC1 . ActivatAlthough TSC2 plays an indispensable role in AMPK inhibition of mTOR, there is an additional mechanism independent of TSC2 between AMPK and the mTOR . It has In this study, CPT could activate TSC2 when activates AMPK, but has no activation of raptor, clarifying that a TSC2-dependent mechanism was triggered by CPT in cancer cells. On the other hand, previously we also found that CPT could generate plenty of ROS in cancer cells, then induce MAPK cascade pathways including activation of MAPK/P38, MAPK/JNK and inhibition of MAPK/ERK , resultiThe mTOR enhances tumorigenesis and has become an attractive therapeutic target in cancer. Both rapamycin, a specific inhibitor of mTORC1, and rapamycin derivatives shows highly effective in treating all types of cancer . HoweverIn this study, we found that CPT inhibition of mTORC1 is via activating AMPK-TSC2 axis rather than other mTORC1-related molecules. It suggested that CPT is a potent AMPK activator and should become a potential anticancer agent in clinic.


Review of how targeting key upstream molecules at the recognition phase of innate immunity exert anti-inflammatory effects; a potential therapeutic regimen for inflammatory diseases. The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross-talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL-1β, and IL-6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by “dual blockade” of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad-acting therapeutic regimen against a number of diseases where innate immunity is improperly activated. The ingenious inflammatory network preserves host integrity and is essential in homeostasis. To fulfill host protection, the immune system needs sensors that respond to potential dangerous inducers, either of exogenous (PAMPs) or endogenous (DAMPs) origin. The instantly acting sensors of innate immunity are the first line of defense by recognizing these conserved molecular structures or patterns. PRRs represent an upstream immunologic hierarchy, sensing danger and subsequently signaling downstream through a multitude of different pathways. PRRs are classified based on their ligands and downstream effector pathways . NeverthAccording to the danger model introduced by Matzinger , the immThe complement system and TLRs are both important upstream components of the innate immune system, recognizing ligands of exogenous and endogenous origin initiating downstream inflammatory effects. These sensors and effector pathways act as partly individual branches but are connected through a considerable cross-talk, representing a vast redundancy of the host defense –7. In 20The present review highlights the significance of complement and TLRs in various inflammatory models and importantly, demonstrates the efficacy of single and combined inhibition of these 2 upstream branches of innate immunity. The results comprise data from different experimental inflammatory ex vivo and in vivo models, including the use of different species. The interpretations of data from the large animal studies can be integrated into a complex context where dynamic relations among physiology, inflammation, thrombogenicity, and specific organs emerge.The complement system was discovered in the 1890s, where Nuttal, Bordet, and others described it as a “lytic system” killing bacteria. The system was first named alexin, and Bordet introduced the term “complement” when he, in 1895, discovered that this was a heat-labile system present in normal human serum and was “complementing” the antibodies in killing the bacteria. Of interest, heat inactivation of serum (56°C for 30 min), which Bordet used to distinguish complement (heat labile) from antibodies (heat stable), is still a routine method used in our daily laboratory work in 2016—“heat-inactivated serum.” Notably, this procedure is by no means specific for complement activity, and caution should be taken when interpreting data from such sera [Immunology Today (now Trends in Immunology) stated the following: “Many immunologists hold that complement is baffling or irrelevant or, most conveniently, both but a recent meeting emphasized that complement is interesting and that it may be important, even only as an elegant model system” [Nearly 100 yr later—in 1984—there was a meeting of The Royal Society  on complement, and the summary in The complement system is built up of multiple proteins organized in 3 separated pathways for broad recognition, with a central C3 convertase for potent response amplification and an effector phase acting instantly upon cleavage of C3 and C5  recognition phase, 2) C3/C3 convertase, and 3) C5/C5a/C5aR1. Molecular targets at the recognition phase include C1s and MASP-2, where blockade is investigated for the treatment of cold agglutinin disease and complement-mediated thrombotic microangiopathy, respectively . FurtherThe level of C3 activation encompasses drugs preventing C3 cleavage by direct-targeting C3. The cyclic peptide compstatin and later-developed acetylated derivatives, including Cp40, inhibit complement activation by binding C3, sterically preventing the binding of the convertase and C3 cleavage  Table 1Table 1. 35Clinical trials, registered in FDA and the European Medicines Agency, using complement-targeted therapeutics, study primarily inflammatory diseases with a clear complement-dependent profile, many caused by mutations in complement genes related to either activation by gain of function or regulation by loss of function, which both lead to an homeostatic imbalance, producing pathologic, increased complement activation . It remaProbably, the best described PRRs are the TLRs, which are up-regulated upon stimulation and found on nearly all cells, especially on cells important in innate immunity signaling, such as dendritic cells, leukocytes, macrophages, and endothelial cells. Ten different human TLRs are known today (TLR1–10), whereas there are 13 known in mice , 41. TLRIt is well established that CD14 enhances LPS responsiveness by binding LPS and facilitating LPS transfer to TLR4–MD2 . In addiSeveral inhibitors, including antibodies or compounds against TLRs and coreceptors, such as MD2 and CD14, have been characterized. However, most of them have only been evaluated in vitro and in rodent models, which makes it difficult to assess their clinical relevance. The main findings from these studies are that TLR inhibition is beneficial in viral infections, SIRS, and sepsis, as well as in cerebral, myocardial, renal, and hepatic I/R injury, but may be detrimental in intestinal I/R injury , 50, 51.TLR inhibition is only sparsely evaluated in large animal models and clinical studies. TLR2 receptor blockade has been evaluated in mice with the mAb OPN-301, which prevents TLR2 dimerization with TLR1 or TLR6 and downstream cytokine production  Table 1Table 1. E. coli infused sheep [Specific inhibition of TLR4 has been tried using several small molecular agents. The small compound TAK-242 (resatorvid) is a direct TLR4 inhibitor, which blocks the intracellular binding of coadaptor proteins to TLR4 and thus, inhibits downstream signaling . Pretreaed sheep , also whed sheep . Howevered sheep .n = 1961) phase III study in severe septic patients [Another TLR4 inhibitor, E5564 (eritoran), is a structural analog of the lipid A portion of LPS, which binds to MD2 and thus, prevents LPS binding and TLR4 activation  Table 1Table 1. patients . Eritorapatients . RecentlE. coli sepsis in pigs [n = 40) revealed inconclusive results, with 1 patient experiencing anaphylaxis upon IC-14 infusion [As mentioned above, CD14 is implicated in activation of several TLRs. It has been shown that a blockade of CD14 attenuates central inflammatory cytokines in plasma and organs and reduces the thrombogeneic state induced by  in pigs , 66. In  in pigs , 68 , chemokines , growth factors , and other ILs  [meningitidis-induced inflammation [d IL-17) . Surprisammation .Although the CD14-driven pathway apparently plays a major role in Gram-negative-induced inflammation, complement plays a comprehensive part too. The complement dependence (C3 and C5) of a vast range of inflammatory responses, as well as the relative importance of CD14 in Gram-negative-induced inflammation, was delineated in blood of a C5-deficient human, nature’s own gene knockout model . It was E. coli [On a transcriptional level, microarray technology revealed a significant change—up- or down-regulation—in >2200 transcripts when human whole blood was challenged with E. coli . As manyaureus in a human whole-blood model demonstrated that simultaneous inhibition of CD14 and complement efficiently reduced the inflammatory response [The effect of complement and CD14 inhibition on Gram-positive bacteria is sparsely evaluated. One study using various strains of Staphylococcus response . HoweverThe pathophysiology of sepsis is complex but can be regarded as a prototype of a host-threatening systemic inflammatory response unleashed by infection. For years, researchers have sought various inhibitory targets to attenuate and get control of the overwhelming and counterproductive sepsis-induced inflammatory response. Clinical studies using mAb toward the central proinflammatory cytokines TNF and IL-1β have failed to improve survival , 80. DroE. coli [N. meningitidis [E. coli-induced sepsis in pigs, combined inhibition of C5 and CD14 essentially abolished the formation of all proinflammatory cytokines, extensively inhibited up-regulation of CD11b on granulocytes, and partly prevented the procoagulant state of sepsis [Pigs are suitable for medical research, as they have closer anatomic, physiologic, and immunologic relations to humans compared with rodents , and addE. coli  or N. mengitidis . Both off sepsis . In addif sepsis . The subf sepsis , 89, butf sepsis .The efficacy of the combined regimen is convincing also in polymicrobial sepsis, a clinically, more relevant model, mimicking abdominal sepsis seen after bowel rupture and anastomotic leak. In mice, combined inhibition of C5 and CD14 profoundly attenuated all inflammatory markers and importantly, significantly increased survival, whereas single inhibition of C5 or CD14 did not . ComparaE. coli-induced inflammation ex vivo [The different sepsis studies demonstrate that inhibition of bottleneck molecules belonging to 2 main systems of recognition has the potential to act broadly on the numerous biomarkers and reveal pronounced effects on downstream mediators and importantly, on clinical outcome. Although the administration of inhibitors before the induction of sepsis is a limitation with these experiments, all studies clearly demonstrate the efficacy of upstream inhibition and belong to the field of science exploring the proof of concepts. In general, the timing of intervention is a great challenge within the field of sepsis; how can we properly identify the patient and provide treatment early enough and not at the time often described as the point of no return? With consideration of the latter, it is therefore interesting and promising that combined postchallenge inhibition of complement and CD14 significantly attenuate central proinflammatory biomarkers in experimental  ex vivo .Today, no studies in big or small animals are published that evaluate combined TLR and complement inhibition in sterile inflammation. Therefore, an extensive area of research needs attention. Meconium, aspirated by term newborns, may induce a life-threatening respiratory condition called meconium aspiration syndrome . ExperimBoth complement and the TLRs are implicated in I/R injury, events that frequently happen in conjunction with infarction, trauma, and transplantation , 98. An The targeting of bottleneck molecules at the level of innate immune recognition of PAMPs and DAMPs may present a particularly efficient way of attenuating the inflammatory response, as all known innate immune systems closely interact. Complement component C3 or preferentially, C5 inhibition, combined with neutralization of the CD14 molecule, fulfills this level of ambition. We suggest that this combined inhibition approach can be a clinically relevant treatment regimen to block an inappropriate and overwhelming inflammatory reaction and restore the homeostasis of the host.A.B.-D., S.E.P., P.H.N., T.E., and T.E.M. wrote the review.The authors declare no conflicts of interest.


The homeodomain protein PDX-1 is a critical regulator of pancreatic development and insulin production in pancreatic β-cells. We have recently shown that PDX-1 is a substrate of protein kinase CK2; a multifunctional protein kinase which is implicated in the regulation of various cellular aspects, such as differentiation, proliferation, and survival. The CK2 phosphorylation site of PDX-1 is located within the binding region of the E3 ubiquitin ligase adaptor protein PCIF1. To study the interaction between PDX-1 and PCIF1 we used immunofluorescence analysis, co-immunoprecipitation, GST-pull-down studies, and proximity ligation assay (PLA). For the analysis of the stability of PDX-1 we performed a cycloheximide chase. We used PDX-1 in its wild-type form as well as phosphomutants of the CK2 phosphorylation site. In pancreatic β-cells PDX-1 binds to PCIF1. The phosphorylation of PDX-1 by CK2 increases the ratio of PCIF1 bound to PDX-1. The stability of PDX-1 is extended in the absence of CK2 phosphorylation. Our results identified protein kinase CK2 as new important modulator of the stability of PDX-1. Protein kinase CK2 is a multifunctional and pleiotropic protein kinase that plays critical roles in cell differentiation, proliferation, and survival ,2,3. CK2Recently, we identified the transcription factor PDX-1 as a new substrate for protein kinase CK2 . The panWe have previously shown that PDX-1 is a substrate for protein kinase CK2 . PhosphoA prerequisite for a physical interaction is the co-localization of two proteins. In a first experiment we performed an in situ immunofluorescence analysis with PDX-1 and PCIF1 specific antibodies in mouse pancreatic MIN6 β-cells. A more refined type of analysis to study the interaction of proteins in situ, is the proximity ligation assay PLA . To anal35S]-methionine labeled PCIF1 in its wild-type form and incubated it with GST-tagged PDX-1 wild-type and the CK2-phosphodeficient (PDX-1T231A/S232A) or the CK2-phosphomimicking (PDX-1T231D/S232E) mutant. GST-tagged PDX-1 and bound PCIF1 were pulled-down with GSH sepharose, complexes were eluted and separated on an SDS polyacrylamide gel. Proteins were stained with Coomassie blue and subjected to autoradiography. The result is shown in T231D/S232E bound considerably more PCIF1. After evaluation of four independent experiments the binding capacity of the D/E mutant was calculated to be 1.5 fold better than for wild-type or the AA mutant  or after 3, 6, 12, or 24 h of treatment. The cell extracts were analyzed on an SDS polyacrylamide gel followed by Western blot with an anti-FLAG-tag-antibody. As shown in T231A/S232A. The experiment was done in triplicate, the resulting bands for PDX-1 were scanned, quantified and normalized to the corresponding α-tubulin loading control. The mean ratios (PDX-1/ α-tubulin) +/- standard deviation of the densitometric quantification are shown in T231A/S232A was about 24 h. Thus, PDX-1, which can no longer be phosphorylated by CK2, is much more stable than the corresponding wild-type form.In the next step we asked whether binding of PDX-1 to PCIF1 might have an influence on the stability of PDX-1. To determine the stability of PDX-1 wild-type, as well as of the CK2 phosphorylation mutant PDX-1S232A/T231A for 48 h. Cells were treated with or without 5 µM MG132, a proteasome inhibitor, for another 11 h and lysed. PDX-1 protein levels were analyzed on a 12.5% SDS polyacrylamide gel and transferred to a PVDF membrane followed by Western blotting using anti-FLAG tag and anti-α-tubulin antibodies.It was shown that PCIF1 and Cullin 3 target PDX-1 for ubiquitination . TherefoAs shown in From these various results we conclude that CK2 phosphorylation of PDX-1 has a massive influence on the stability of PDX-1 and this phosphorylation confers susceptibility of PDX-1 protein to proteasomal degradation.Protein kinase CK2 is classified as a serine/threonine kinase with more than 400 different substrates, which indicates the pleiotropic functions of CK2 . By usinThe stability of some proteins, such as myc , ataxin-229AVTS232 which is quite similar to the SRC-3 binding motif of PCIF1. The threonine and serine residue within this consensus motif are the phosphorylation sites for protein kinase CK2. We found that the binding of PDX-1 to PCIF1 was enhanced upon using phospho-mimicking mutants where T231 and S232 were exchanged by an acid residue; on the other hand, PDX-1 binds weaker to PCIF1 upon inhibition of CK2. There are several examples that binding of E3 ubiquitin ligases, like β-TRCP or FBW7, to their substrates is modulated by phosphorylation -phosphate into the synthetic CK2 specific substrate peptide with the sequence RRRDDDSDDD γ ATP. The mixture was spotted onto a P81 ion exchange paper. The paper was washed three times with 85 mM H3PO4. After treatment with ethanol, the paper was dried and the Čerenkov radiation was determined in a scintillation counter.To determine the activity of CK2 after its inhibition, cells were treated with CX-4945 or left untreated, lysed, and the extracts were used in a kinase filter assay. In this assay we measured the incorporation rate of [RDDDSDDD . Twenty


Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulation of ANXA1 functions has been associated with tumorigenesis and metastasis events in several types of cancer. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized that ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells.Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins.We identified a total of 8110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins had ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network proposing new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and suggested closely coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of calmodulin-dependent protein kinase II (CAMK2), P21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and IκB kinase (IKK) upon loss of ANXA1. Integrative analysis revealed regulation of the WNT and Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is downregulation of transcriptional effects of TEA domain family (TEAD) suggestive of ANXA1-responsive transcriptional rewiring.The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and highlighted its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.The online version of this article (doi:10.1186/s13058-017-0924-4) contains supplementary material, which is available to authorized users. Annexin-1 (ANXA1), also known as lipocortin-1, belongs to a multigene superfamily of calcium-dependent phospholipid binding proteins that regulate inflammatory responses . ANXA1 i+/- and ANXA1-/- murine mammary gland cells to understand the ANXA1-modulated signaling network upon breast cancer initiation. Our phosphorylation dataset complements our previous proteome profiling, and provides novel insights into several ANXA1-dependent processes including its role in rewiring adhesion-related machinery.Besides the inflammatory effects, ANXA1 also plays essential roles in cell proliferation, differentiation, apoptosis and cancer . By modu+/- and ANXA1-/- lysates.Stable isotope labeling with amino acids in cell culture (SILAC)-adapted cell lines previously established from mammary gland epithelial cells from ANXA1-heterozygous and ANXA1-deficient mice were used in this study . The SILFilter-aided sample preparation (FASP) was performed as described with slight modifications . In shor2 10 μm . After incubating the mixture for 10 minutes at room temperature, the sample was centrifuged at 4000 g for 3 s. The supernatant was collected and the above process was repeated with another portion of the beads. The beads were washed four times with 1 M glycolic acid/80% acetonitrile (v/v)/2% TFA (v/v) solution and four times with 80% acetonitrile (v/v)/0.1% TFA (v/v) solution after transferring to a 200-μL pipet tip plugged with two layers of C8 empore disks (3 M Empore). Finally, the phosphopeptides were eluted using 500 uL 40% acetonitrile (v/v) containing 15% NH4OH (m/v) followed by vacuum-concentration to ∼ 40 μL.Phosphopeptides were enriched from the tryptic peptides as described with slight modifications . Briefly3COOH, 20 mM H3PO4, 20 mM H3BO3 and NaOH, pH 11. The sample was then fractionated on a micropipette tip with six layers of 3 M Empore Anion Exchange disks  stacked into it. Peptides were loaded at pH 11 and fractions were subsequently eluted with buffer solutions of pH 8, 6, 5, 4 and 3, respectively into their respective C18 stage tips.Phosphopeptides were fractionated as described, with slight modifications . Brieflyr = 60,000 at m/z 400, an AGC target of 1e6, and a maximum injection time of 500 ms. For each scan, the top ten most intense peptide ions with intensity threshold >2000 and charge state ≥2 were isolated sequentially to a target value of 1e4, and the precursor ions were fragmented by collision-induced dissociation in a linear ion trap using normalized collision energy of 35%. Dynamic exclusion was applied for 30 s using a maximum exclusion list of 500.Full-scan MS spectra corresponding to m/z 300–1400 were acquired using Orbitrap or Orbitrap XL (Thermo Fisher) for all samples with a resolution of Data were processed using MaxQuant (Version 1.3.0.5) , 26 agaiz score threshold of 2 or less than -2 (approximately equating to at least 1.8-fold change in this dataset) reliably in three out of four experiments were considered to be regulated. Functional enrichment for the regulated phosphoproteins was obtained as Gene Ontology (GO) terms corresponding to biological process, cellular component, and molecular function using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool [p value <0.01 in either of the increased or decreased phosphorylation groups were visualized as a heatmap.For differential expression analysis only those phosphosites that can be localized with a localization probability >0.75 were considered. The SILAC ratios of the corresponding peptides were log-transformed, and each replicate was median-centered and scaled for median absolute deviation (MAD). Only those phosphopeptides greater than the robust ion tool . Only thThe upstream kinases of the regulated phosphosites were obtained by mapping the site-specific information against a comprehensive knowledge base of experimentally derived kinase-substrate relationships (KSR) available from KSR-LIVE . The intz statistic. The z score was converted to a p value and was FDR-adjusted in R environment.Upstream kinase prediction was also carried out using the NetworKIN (version 3.0) algorithm that uses kinase consensus motif along with contextual modeling for predicting KSRs . The moup value <0.05) in at least one of the groups were retained for hierarchical clustering and visualization as a heatmap. To perform gene-set enrichment analysis a pre-ranked list based on average fold change derived (ANXA1-/-/ANXA1+/- ratio) from both proteome and phosphoproteome data was created. For genes with multiple phosphopeptides, fold changes corresponding to the most regulated phosphopeptides (highest or lowest fold change) were used for ranking. For the others that did not meet the previously set robust z score cutoff, those phosphopeptides with the highest ratio were used. Gene-set enrichment analysis (GSEA) was performed using all curated mouse GO biological processes and pathways individually for both the proteome and phosphoproteome data [p value <0.01 were visualized as a network using the Enrichment Map plugin as implemented in Cytoscape [From the proteome data, those proteins that were 2.5 MAD away from the median (roughly equating to twofold change) in three out of four experiments were considered as regulated. For comparative analysis, proteins that displayed change in abundance at both phosphoproteome and proteome level, and those that displayed changes only in the phosphoproteome level were grouped into four distinct clusters. Functional enrichment for the cellular component was carried out for each of the clusters and the significantly enriched clusters  were visualized using the Treemap package from R. For construction of the interaction network we used protein-protein interactions curated in the InWeb_InBioMap database that has 2.8 times more interactions than other comparable resources and is also functionally superior [Expression-driven transcription factor prediction analysis was carried out for all differentially regulated proteins in the proteome dataset using the Transcription Factor database (TRANSFAC) . The anasuperior . The idesuperior .+/− and ANXA1−/− murine mammary gland epithelial cells implicated a role for ANXA1 in breast cancer initiation and progression [+/− and ANXA1−/− murine mammary cells as depicted in Fig.-/- cells versus light ANXA1+/- cells) and reverse experiments (heavy ANXA1+/- cells versus light ANXA1-/- cells) identified a total of 11,235 unique phosphopeptides carrying 8110 unique phosphorylation sites on 2626 proteins  we identified 362 phosphosites on 224 proteins with increased phosphorylation and 220 sites on 153 proteins with decreased phosphorylation in three out of four experiments -associated kinases such as MKNK2, MAP3K8 and RPS6KA3 and microtubule regulatory kinases such as DCLK1, MAST2 and MARK3 are among those with induced phosphorylation. This is interesting as some of these kinases have a profound influence on cell migration activities of pathways including WNT and NF-κB signaling. Enhanced phosphorylation of serine/threonine kinase receptor TGFBR2 on multiple serine sites at Ser573, Ser576 and Ser578 was also observed in ANXA1activity . Conversactivity , 39. Foractivity . Among aNext, we looked into ontology-based enrichment for those modulated phosphoproteins Fig. . It becaProteins with downregulated sites in ANXA1-deficient mammary epithelial cells displayed preferential enrichment for microtubule-based processes and ATP-dependent chromatin remodeling. A few phosphoproteins with reduced site abundance were also enriched for actin cytoskeletal processes and localized to focal adhesions indicative of a complex network of phosphorylation-mediated events that establish cell movement. While the role of ANXA1 in regulating the actin cytoskeleton has been reported earlier, our dataset exposes its possible novel associations in influencing microtubule-filament-based processes. We uncovered reduced phosphosite abundance on microtubule motor proteins including kinesin and dynein, and on other microtubule-associated proteins such as MAP2 and CLIP2. It is worthy to note that one of the key proteins that influence microtubule stability, stathmin (STMN1) was found with increased phosphosite abundance at the functional site Ser16 that inhibits microtubule depolymerization in ANXA1-deficient cells, highlighting the pervasive role of ANXA1 in regulating microtubule dynamics .Next, we asked how the functions of those proteins that changed in total or site abundance compared with each other in ANXA1-deficient mammary cells . Of all the regulated sites, upstream kinase was experimentally curated only for 56 phosphosites on 45 proteins. The interconnectivity of the kinome network Fig.  was obtaThough the kinome network highlighted KSRs from diverse kinase families, it is not representative of the entire signaling network as many of our regulated phosphosites did not have experimentally curated upstream kinase. Hence, we additionally predicted KSRs for all regulated phosphoproteins using the NetworKIN algorithm that assigns kinases based on linear consensus motifs and network proximity . We perfp value <0.05) that were potentially regulated in the absence of ANXA1 Additional file 2: Figure S1.A Distribution of Andromeda scores of the identified phosphorylated peptides show that most peptides were identified with high scores with a median score of 130. B Distribution of localization probabilities of identified phosphorylation sites. The median probability was ~ 0.97. C Assessment of reproducibility between the four biological replicates carried out with SILAC label swapping shows optimal reproducibility among the forward and reverse experiments. The correlation scores are indicated in each scatter plot. (PDF 1803 kb)Phosphoproteomic data quality and reproducibility. Additional file 3: Figure S2.A Distribution of robust z scores of the quantified phosphopeptides. The regions highlighted in red and blue correspond to upregulated and downregulated phosphopeptides, respectively. The sites that were reliably regulated above or below the MAD threshold of ± 2 in three out of four replicates were considered ANXA1-responsive and these at least displayed a 1.8-fold change. The robust z scores were based on log2 normalized fold changes. B Distribution of serine, threonine and tyrosine sites among the regulated phosphorylation sites. (PDF 1059 kb)ANXA1-regulated phosphoproteome in mammary epithelial cells. Additional file 4: Table S2.List of ANXA1-responsive phosphorylation sites in mammary epithelial cells from ANXA1-heterozygous and ANXA1-deficient mice. (XLSX 1001 kb)Additional file 5: Figure S3.A Number of class I phosphorylation sites with corresponding protein quantification. Except for 1550 sites on 765 proteins that had no corresponding protein measure, the rest of the sites mapped to 1765 proteins with abundance measures. B Intensity-based density plot comparing protein and phosphorylation abundance shows poor correlation. (PDF 1234 kb)Comparison of quantified proteome and phosphoproteome in ANXA1-deficient mammary epithelial cells. Additional file 6: Table S3.List of all identified proteins in mammary epithelial cells from ANXA1-heterozygous and ANXA1-deficient mice. (XLSX 11902 kb)Additional file 7: Table S4.Site-specific functions of ANXA1-regulated phosphorylation sites. (XLSX 23 kb)Additional file 8: Table S5.Cellular component enrichment of ANXA1-modulated phosphoproteins. (XLSX 10 kb)Additional file 9: Figure S4.z scores of the quantified proteins. The regions highlighted in orange and blue correspond to upregulated and downregulated phosphopeptides, respectively. Only those proteins regulated in at least three out of four experiments were considered regulated. (PDF 786 kb)ANXA1-regulated proteins in mammary epithelial cells. Distribution of robust Additional file 10: Table S6.List of all ANXA1-regulated proteins in mammary epithelial cells from ANXA1-heterozygous and ANXA1-deficient mice. (XLSX 819 kb)Additional file 11: Table S7.Enrichment of cellular component terms among the different categories. (XLSX 12 kb)Additional file 12: Table S8.Summary of associated pathways and localizations of ANXA1-responsive phosphoproteins. (XLSX 29 kb)Additional file 13: Figure S5.Clusters enriched in ANXA1-regulated protein interaction network. Integrated protein-protein interaction network was constructed using those proteins with ANXA1-responsive phosphorylation changes along with transcription factors predicted from ANXA1-regulated proteome. Clusters were identified using GLay community structure detection and the top clusters identified along with their associated functions are shown. (PDF 5678 kb)Additional file 14: Table S9.Migration-associated ANXA1-responsive phosphoproteins. (XLSX 43 kb)


SU6656, a Src kinase inhibitor, was reported to increase fat oxidation and reduce body weight in mice, with proposed mechanisms involving AMP-activated protein kinase (AMPK) activation via inhibition of phosphorylation of either LKB1 or AMPK by the Src kinase, Fyn. However, we report that AMPK activation by SU6656 is independent of Src kinases or tyrosine phosphorylation of LKB1 or AMPK and is not due to decreased cellular energy status or binding at the ADaM site on AMPK. SU6656 is a potent AMPK inhibitor, yet binding at the catalytic site paradoxically promotes phosphorylation of Thr172 by LKB1. This would enhance phosphorylation of downstream targets provided the lifetime of Thr172 phosphorylation was sufficient to allow dissociation of the inhibitor and subsequent catalysis prior to its dephosphorylation. By contrast, sorafenib, a kinase inhibitor in clinical use, activates AMPK indirectly by inhibiting mitochondrial metabolism and increasing cellular AMP:ADP and/or ADP:ATP ratios.  •SU6656 activates AMPK independently of the Src kinases Src, Yes, and Fyn•SU6656 acutely inhibits AMPK by competing with ATP at the active site•SU6656 paradoxically activates AMPK by enhancing phosphorylation by LKB1•By contrast, sorafenib activates AMPK indirectly by increasing cellular AMP/ADP The kinase inhibitors SU6656 and sorafenib paradoxically activate AMPK. Ross et al. show that SU6656 inhibits AMPK by binding directly at the catalytic site, yet promotes phosphorylation and activation by LKB1, while sorafenib activates AMPK indirectly by inhibiting mitochondrial function. It exists in eukaryotes as heterotrimeric complexes comprising catalytic α and regulatory β and γ subunits , occurri, CaMKK2 . The AMP, CaMKK2 , where a, CaMKK2 . The γ s, CaMKK2 . Binding, CaMKK2 , while b, CaMKK2 . AMPK isMost studies suggest that LKB1 complexes are constitutively active, with Thr172 phosphorylation being regulated instead by binding of adenine nucleotides to AMPK . HoweverWe now confirm that SU6656 activates AMPK but show that it does this not by modulating tyrosine phosphorylation of LKB1 or AMPK but, surprisingly, by direct interaction with the AMPK kinase domain. Recently sorafenib, another kinase inhibitor already licensed for treatment of some cancers , was repTreatment of HEK293 cells with increasing concentrations of SU6656 caused activation of AMPK that correlated with Thr172 phosphorylation; it also increased phosphorylation of one downstream target, ACC, but not another, Raptor. Another AMPK activator, berberine, caused a much smaller degree of AMPK activation and Thr172 phosphorylation, yet had similar effects on ACC phosphorylation and did promote Raptor phosphorylation A. By immIf SU6656 acts by blocking phosphorylation of LKB1 by Fyn and promoting migration of LKB1 to the cytoplasm , the AMPThe results in We next transiently transfected DNAs encoding GFP, or GFP-tagged wild-type (WT), Y261F/Y365F (FF), or Y36F/Y261F/Y365F (FFF) mutants of LKB1 into HeLa cells, which lack endogenous LKB1. AMPK was activated C and pho2+ and activates CaMKK2) all activated AMPK, and these responses were unaffected by the Y436F mutation  mutant . As expeWe also examined the effects of sorafenib, another kinase inhibitor reported to paradoxically activate AMPK . Sorafen50] = 1.0 ± 0.1 μM [±SEM]). The single K40A or K42A mutations exhibited greatly reduced activation by A769662, while the double (AA) mutation abolished it (50 = 23 ± 4 μM) .We next tested whether SU6656 activates AMPK by binding at the ADaM site. Another recently described activator, MT 63-78, may bind this site since, like A769662 and salicylate, it is a more potent activator of AMPK complexes containing β1 rather than β2 . In crysished it C. Simila ± 4 μM) D. By con ± 4 μM) E. AlthouWe next expressed the FLAG-tagged WT or AA mutant of AMPK-α1 by transient transfection in HEK293 cells, treated with various agents, and measured AMPK activity in anti-FLAG immunoprecipitates. In ATP = 151 ± 7 μM, and KiSU6656 = 0.17 ± 1 μM.Since SU6656 activation did not require functional γ-subunit or ADaM sites, this left the catalytic site as the most likely binding site. Indeed, SU6656 inhibits AMPK as effectively as Src . To exam50 = 0.22 ± 0.02 μM), while inhibition was almost 10-fold less potent with the AT mutant (IC50 = 1.7 ± 0.3 μM).In a crystal structure of the human α2β1γ1 complex , the sid50 for inhibition of catalytic activity (0.22 ± 0.02 μM).We next used unphosphorylated α2-KD and tested the effect of SU6656 on its activation and phosphorylation by LKB1. Concentrations of SU6656 up to 10 μM promoted activation of WT α2-KD by LKB1 by 1.9 ± 0.2-fold and increased Thr172 phosphorylation D and 5E,To test whether SU6656 binding inhibited Thr172 dephosphorylation, we used bacterially expressed α2-KD or a full-length α2β2γ1 complex, both of which had been phosphorylated using CaMKK2. We also expressed FLAG-tagged AMPK-γ1 in HeLa cells, either the WT or an R299G (RG) mutation , which prevents AMP binding at the critical site 3 . The AMP2+ and activates the alternate upstream kinase CaMKK2. By contrast, three activators that work by increasing cellular AMP/ADP  all failed to activate or cause Thr172 phosphorylation of the α1-KD while doing this with the endogenous complexes, consistent with the fact that only the latter have AMP/ADP binding sites on the γ subunit. A769662 is primarily an allosteric activator  by transient transfection in HEK293 cells, treated the cells with various AMPK activators, and then assayed both the expressed α1-KD (precipitated using anti-FLAG) and endogenous full-length α1 complexes (precipitated using anti-α1 antibodies recognizing an epitope outside the α1-KD). SU6656 increased kinase activity A and Thrctivator  and onlyctivator . AlthougThese results showed that only the kinase domain was required for SU6656 to activate AMPK in intact cells. To confirm that it acted by binding at the catalytic site, we expressed FLAG-tagged full-length AMPK-α1 either as the WT or the AT mutant. www.kinase-screen.mrc.ac.uk/kinase-inhibitors), so increased Thr172 phosphorylation by LKB1 in intact cells is not observed. By searching this database, we found one other inhibitor, MRT199665, where inhibition of LKB1 was less potent than that of AMPK, as with SU6656. When HEK293 cells were incubated with increasing concentrations of MRT199665, we observed a modest increase in AMPK activity and Thr172 phosphorylation, more than with A769662 but much less than with phenformin or SU6656. However, unlike SU6656, A769662, or phenformin, MRT199665 led to inhibition of phosphorylation of the downstream target ACC .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Grahame Hardie (SYF cells were from the ATCC and were used within 3 months of purchase. Human cells (HEK-293 and HeLa) were from the European Collection of Cell Cultures (ECACC) and were re-validated by STR profiling by Public Health England prior to the project (certificate dated 08/14/2015). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.Kpn1 and 3' Xho1 sites, the resulting PCR product was cloned into pcDNA5 FRT. A plasmid encoding the AMPK-α2 kinase domain (α2-KD) was made by amplifying residues 1-310 with 5' BamH1 and 3' Xho1 sites, the resulting PCR product was cloned into pGEX6P2. Point mutations were generated using the Quikchange II site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing.A plasmid encoding the AMPK-α1 kinase domain (α1-KD) was made by amplifying residues 1-312 with an N-terminal FLAG-tag and 5' -1) prior to addition of compounds under test. Transient transfections were carried out 36-48 hr prior to experiments, using Fugene 6 according to manufacturers’ instructions.HEK-293 cells stably expressing tetracycline-inducible FLAG-tagged human AMPK-γ2, or an R531G mutant, were generated by introducing the corresponding DNAs into Flp-In™ T-REx™ 293 cells . ExpressSemi-preparative HPLC see  was perf1H NMR  δ 13.15 , 11.30 , 8.05 , 8.0 , 7.54 , 7.13 , 6.76 , 2.80 , 2.66 , 2.60 , 1.86 , 1.78. Material purified by semi-preparative HPLC gave an identical spectrum.NMR spectra were recorded on Bruker AVANCE II 500 spectrometer. Splitting patterns of spectral multiplets are indicated as s, singlet; br s, broad singlet; d, doublet; dd doublet of doublets; m, multiplet. Analysis of SU 6656 supplied by Sigma: +) of 372.4 (expected mass for C19H21N3O3S = 371.46), as well as a [2M+H]+ peak of 743.3 (not shown).LC-MS analysis was performed using a Dionex UltiMate3000 series LC instrument with a Waters Xbridge C18 column  at a flow rate 0.3 mL/min. A linear gradient from 5%-95 % buffer B in buffer A over 8 min was used for elution . The LC was connected to a Bruker MicroTOF and MS was acquired in ESI positive mode. The major peak detected by UV absorbance at 254 nm generated a m/z value  δ 8.28 , 8.15 , 8.08 , 7.95-7.84 , 7.39-7.36 , 6.86 ; 13C NMR  δ 155.3, 140.5, 135.7, 135.2, 133.8, 133.0, 131.6, 128.8, 128.1, 127.5, 123.7, 117.4, 116.8, 116.5, 113.1, 107.4, 85.9.MT 63-78 was synthesized as described previously . LC:MS wAMARA peptide  and assayed at 200 μM ATP  using th peptide  as subst peptide A where t2 mM ATP . The AMP2 mM ATP  in lysat2 mM ATP . LKB1 wa2 mM ATP .2 ± a limiting amount of LKB1:STRAD:MO25 complex (0.7 μg), in the presence or absence of AMP (200 μM) or SU6656 (3 μM) as indicated. The reaction was terminated by addition of 1 ml of ice-cold IP buffer  Triton X-100, 0.1 mM phenylmethane sulphonyl fluoride, 5 μg/ml soybean trypsin inhibitor, 1 mM benzamidine, and after an additional wash in the same buffer the AMPK activity or Thr172 phosphorylation status was determined by kinase assay or Western blotting.Plasmids encoding FLAG-tagged AMPK- γ1 (WT or RG mutant) were transfected into HeLa cells using Fugene 6. Cell lysate (90 μg protein) was immunoprecitated using EZview Red anti-FLAG M2 affinity gel, and incubated for 10 min in a total volume of 75 μl with 200 μM ATP, 5 mM MgCl2 (9.8 mM) to activate PP2Cα; reactions were conducted in a shaking incubator at 30°C for 10 min and were stopped by diluting 5 μl into 300 μl Hepes buffer (as above). An aliquot (5 μl) was then taken to assay AMPK.Active human AMPK (α1β1γ1 complex) phosphorylated on Thr172 was generated as described previously . DephospThe levels of AMP, ADP and ATP were measured using a TSQ Quantiva interfaced with Ultimate 3000 Liquid Chromatography system (ThermoScientific), equipped with a porous graphitic carbon column . Mobile phase buffer A consisted of 0.3% (v/v) formic acid adjusted to pH 9 with ammonia prior to a 1:10 dilution. Mobile phase buffer B was 80% (v/v) acetonitrile. The column was maintained at a controlled temperature of 30°C and was equilibrated with 10% buffer B for 5 minutes at a constant flow rate of 0.06 mL/min. Aliquots of 1 μL of each sample were loaded onto the column and compounds eluted with a linear gradient of 10%-60% buffer B over 9 min. Buffer B was then increased to 100% within 1 min, and the column washed for 5 min with 100% Buffer B. Eluents were sprayed into the TSQ Quantiva using Ion Max NG ion source with ion transfer tube temperature set to 350°C and vaporizer temperature 125°C. The TSQ Quantiva was run in negative mode with a spray voltage of 2600, sheath gas 40 and Aux gas 10. Levels of ATP, ADP and AMP were measured using multiple reactions monitoring mode (MRM) with optimised collision energies and radio frequencies previously determined by infusing pure compounds. Three transitions were used to monitor each of the three compounds, ATP , ADP  and AMP .Cellular oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyser according to manufacturers’ instructions, as specified previously .Data were fitted to the specified equations by non-linear regression using GraphPad Prism 6 for Mac OSX.E. coli was identified by peptide mass fingerprinting.SDS-PAGE was performed using precast Bis-Tris 4–12% gradient polyacrylamide gels in the MOPS buffer system (ThermoFisher Scientific). Proteins were transferred to nitrocellulose membranes using the iBlot 2 system (ThermoFisher Scientific). Membranes were blocked for 1 hr in Li-Cor Odyssey blocking buffer. The membranes were probed with appropriate antibody (0.1–1 μg/ml) in TBS-Tween and 2% (w/v) non-fat dried skimmed milk except where the blotting enhancement system was used (as per manufacturers’ instructions). Detection was performed using secondary antibody (1 μg/ml) coupled to IR 680 or IR 800 dye, and the membranes were scanned using the LICOR Odyssey IR imager. Protein concentrations were determined by Coomassie Blue binding with bovine serum albumin as standard . The ArnNumbers of replicates and statistical significance is indicated in Figures or Figure legends. Numbers of replicates (n) refer to biological replicates, i.e. the number of independent cell cultures analyzed or independent cell-free assays conducted. Significances of differences were estimated with GraphPad Prism 6 for Mac OSX, using 1-way or 2-way ANOVA as appropriate, and (unless stated otherwise) Sidak’s multiple comparison test. Significant differences are indicated either by asterisks: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, or by daggers: †P<0.05, ††P<0.01, †††P<0.001, ††††P<0.0001.F.A.R., S.A.H., F.R.A., and G.J.G. designed and conducted experiments; A.A. and D.J.L. devised a new analytic method and conducted analyses; D.G.H. supervised the project and wrote the first draft of the manuscript; all authors read and amended the manuscript.


Mast cells and basophils depend on aggregation of the high-affinity IgE receptor, FceRI, to initiate secretion. A variety of studies have shown that FceRI densities vary 100 fold among subjects’ basophils and it has been speculated that high densities might be responsible for unusual behaviors of the cells, notably sensitivity to certain monomeric IgE antibodies or spontaneous release. These studies experimentally examined the density dependence of spontaneous release and signaling element expression in subjects’ basophils with FceRI densities ranging from approximately 6000 to 600,000 per cell. Through the use of numerical simulation, this study examined the expectations for spontaneous receptor aggregation and aggregate persistence at densities of FceRI ranging from 5000 to 500,000. Experimentally, FceRI density was not associated with greater spontaneous histamine release even when secretion was enhanced by the inclusion of deuterium oxide in the buffers. There was also no association of 15 activating or de-activating signaling elements with FceRI density. The numerical simulations demonstrated that at densities of ≈500,000 receptors, 13% of receptors were involved in transient aggregates at any given moment but that these aggregates rarely persisted for greater than 10 milliseconds. In contrast, a weak linear antigen aggregator, with ligand affinities known to induce secretion, would generate aggregates persisting for an average of ≈60 milliseconds. These results suggest that although a high density of FceRI likely produces a large number of transient aggregates, these aggregates do not persist long enough to induce signaling that results in secretion and do not induce the cells to alter their expression of several signaling elements known to be important in regulating secretion from human basophils. The results set some boundaries on the aggregation requirements for inducing histamine release from human basophils. Allergic reactions result from the IgE-mediated (immunoglobulin E) activation of mast cells and basophils. These cells express high affinity receptor for IgE (FceRI) and bound IgE confers on these cells the ability to sense the antigenic environment of the cells. A remarkable feature of FceRI expression on basophils and mast cells is the range of possible expression. After synthesis and placement on the cell surface, the residency time of the unoccupied receptor is relatively short; 50% are returned to the inside of the cell every 24 hours. Some of these might be recycled to the cell surface but many are degraded. However, as near as can be determined, if FceRI binds IgE, it will never leave the cell surface, provided aggregation doesn’t occur. This relationship between IgE and the stability of expressed FceRI produces some remarkable levels of FceRI expression. The steady state density of FceRI in the absence of IgE is approximately 5000–10000 per cell (a number which is dependent on the synthetic rate) . It is nRegarding whether the spontaneous aggregates might matter, there are a variety of experimental results that provide some context for the question. Another property of FceRI biology is that while some form of aggregation is required to initiate signaling, it doesn’t have to be a physical crosslink –8. SimplThe question of transience is a harder to answer but two experimental observations provide some insight. First, the signaling cascade actually has differential sensitivity to the transience of the aggregate depending on the readout of activation being assessed. Notably, extremely weak crosslinkers can still drive a signaling reaction that results in gene transcription while not leading to degranulation . A seconTherefore, if the juxtaposition of FceRI molecules need not be very long or close, the question becomes, how dense do the receptors need to be to initiate spontaneous signaling? This issue may have been inadvertently addressed in studies purporting to show that monomeric IgE can induce secretion . Many suMethodology for answering the question of transience experimentally has not been developed. An alternative approach, an aggregation simulation model, was used to explore the potential for long duration spontaneous aggregates.Leukapheresis donors are approved by protocols obtained by the leukapheresis center and the cells used for purification of basophils are considered a residual waste product. The protocol for obtaining blood from the subjects that were not processed with leukapheresis was approved by the Johns Hopkins IRB and the donors provided a written approval of a consent approved by the same IRB. This specific study was encompassed by the above approvals. Written consents are saved as part of the standard record keeping for IRB-approved studies.The following were purchased: PIPES , bovine serum albumin (BSA), EGTA, EDTA, D-glucose, NaF, Na4P2O7, Na3VO4, 2-ME, NP-40, FMLP ; crystallized human serum albumin (HSA) ; fetal calf serum (FCS) and RPMI 1640 containing 25 mM HEPES and L-glutamine ; Percoll, ; Goat anti-human IgE Ab was prepared as previously described .PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2. PAG-EDTA consisted of PAG supplemented with 4 mM EDTA .Blood was partially enriched for basophils using single-step Percoll gradients. After washing to obtain a mononuclear cell preparation, cells were resuspended PAGCM buffer (to assess histamine release). Total histamine content was obtained by lysis of the cell preparation with 1.6% perchloric acid. These experiments included two types of spontaneous release assessments. The standard measurement is in PAGCM buffer without stimulus but a second measurement included 44% deuterium oxide to replace the water in the PAGCM. D2O (deuterium oxide) is known to enhance secretion of any type of stimulation . HistamiHistamine was measured by automated fluorimetry . The perOn a separate occasion, basophils from the same subjects were analyzed for cell surface total IgE using acetate elution. An aliquot of cells was used to determine the total basophil count by alcian blue staining and the remaining cells were treated with acetate buffer, pH 3.7, to dissociate endogenous IgE. The recovered supernatant was neutralized to pH 7 and IgE concentration measured by a total IgE assay (immunoCap). A calculation of the number of IgE molecules present and the cell number allowed the cell surface IgE density to be determined . As descBasophils were purified from leukapheresis packs or from blood obtained by venipuncture. When used at high purity, they were purified to near homogeneity by sequential application of Percoll gradients, countercurrent elutriation and negative selection using the basophil purification kit  and columns from Miltenyi Biotec  . The aveThese methods and a presentation of example blots from these studies have been previously published . All samTo make predictions about the number and duration of spontaneous aggregates, a simulation of IgE/receptor associations with or without physical linkers was developed. The supporting information contains a detailed description of the methodology as appendix 1 and the relevant code framework file  for the It is reasonable to suggest that the resting mechanisms of regulation of secretion may need to track with the receptor density in order to not allow spontaneous, non antigen-induced, secretion. A starting point for determining if receptor expression has an impact on function was to determine whether there was spontaneous secretion that was dependent on the density of FceRI. This was done in 4 separate surveys. In two of these surveys, spontaneous secretion was assessed in normal buffers and in buffer replacing 44% of the water with deuterium oxide. This substitution is known to significantly augment secretion, even without stimulation . Fig 2 sIf spontaneous secretion is not dependent on receptor density, there may be changes in signaling elements that maintain a quiescent state. A first look at this issue was whether peripheral blood basophils (PBB) cultured in IL-3 or CD34-derived basophils (CD34B) cultured in IL-3 and assessed for changes in signaling species show changes related to changes in FceRI. Two kinases that can be shown to increase the signaling and secretion of basophils are the src-family kinase, lyn, and syk . HoweverA survey of the natural distribution of several signaling elements in subjects’ basophils across a spectrum of IgE densities did not reveal marked differences at the high and low end of the density spectrum for FceRI. It is also possible to derive a small amount of data from recent studies examining the change in FceRI expression during treatment of patients with omalizumab. Only 4 signaling species have been examined in peripheral blood basophils, syk, lyn, c-cbl, and cbp/PAG , an interesting issue was raised regarding the impact of high densities of FceRI on spontaneous activation of basophils or mast cells. With a 100-fold range of FceRI expression, it would be surprising if the cells didn’t accommodate the signaling apparatus for conditions that might lead to a great deal of spontaneous aggregation. One can only speculate on the precise reason basophils and mast cells accumulate so many FceRI. One plausible explanation is to prevent a parasite from inducing nonspecific IgE as a defense against specific IgE-mediated secretion as part of the host rejection mechanism. Whatever the reason, the large up-regulation presents a potential problem for the secretory apparatus.The simulations demonstrate that at the highest densities of FceRI that are observed in individuals with high IgE levels, there are a considerable number of spontaneous aggregates present. As noted previously, a variety of studies have suggested that juxtaposition need not be close  but it dThe experimental findings show that there was no relationship between FceRI density and spontaneous histamine release ± deuterium oxide. In other studies we have shown that there is also no relationship between cellular sensitivity  or maximum histamine release . In otheThe observation that there is no relationship between FceRI density and spontaneous release might suggest that the cell uses counter-balancing mechanisms to keep activation in check. One way to counter-balance the increase in receptor is to down-regulate activating signaling steps. Alternatively, up-regulation of termination mechanisms could suppress spontaneous activation. We found no evidence for either of these changes in the molecular species that have been studied in basophils and shown to be participatory during IgE-mediated activation and de-activation. However, the list is short and there remain many other signaling species that would need examination.In addition, there is no a priori reason that any single signaling element might show a correlation with FceRI density. It may be that the entire signal element network adjusts such that only small increments of change occur for any one element. With this in mind, a simple calculation was performed on the data set used for the results in In a limited pilot study of 2 patients treated with omalizumab and 2 treated with placebo, there were no notable changes in the transciptome of basophils that experienced both down-regulated FceRI during treatment and up-regulated syk expression relative to the placebo-treated group (unpublished results). Notably, there were no changes in mRNA expression for a wide variety of expressed signaling species. What is notable from studies of omalizumab treatment is that omalizumab does down-regulate FceRI expression while up-regulating syk expression . This woEven though the persistence of spontaneous aggregates is short, there are approximately 100,000 of them at any given moment on basophils with 500,000 receptors. It remains somewhat surprising that there is no notable consequence to this situation. Perhaps the fact that FceRI has no intrinsic enzymatic activity allows the system to behave this way . It woulS1 FileDescription of the aggregation simulation.(PDF)Click here for additional data file.S2 FileExcel spreadsheet.(XLSX)Click here for additional data file.S3 FileExcel spreadsheet.(XLSX)Click here for additional data file.S4 FileStandard C code; main and model code without user interface code. To relate line numbers in the appendix to the code in this file, a code editor is needed.(C)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigEach IgE molecule was tracked for its association with an aggregate and marked for size of the aggregate at each cycle. A record for all IgEs at each cycle was stored and later scanned for each IgE and its status with respect to aggregate size. If it was marked as in an aggregate of size ≥2 (the gray zone in (TIF)Click here for additional data file.S3 FigPanel A; results of simulation for a 13.5 (filled circles) and 20.0 (filled squares) nm center-to-center separation distance being counted as an aggregate without regard to size or persistence at a given moment. Panel B; relationship between the size of the spontaneous aggregate, the number of aggregates and density of FceRI. The numbers on the ordinate reflect the total number of aggregates .(TIF)Click here for additional data file.S4 FigPersistence for clusters/aggregates of any size larger than monomers for two center-to-center separation distances . The count is for the number of such clusters for the first 75 msec of the simulation at a total receptor density of 270,000.(TIF)Click here for additional data file.S5 Fig6 M-1 was used for the simulation.A single-site affinity of 5x10(TIF)Click here for additional data file.S6 Fig−7 M) of crosslinker (black circles), or suboptimal crosslinker (2.5x10-10 M) (green open diamonds). The histograms on the right edge of the plot show the remaining number of aggregates at times > 75 msecs. Panel B, 3 simulations, at two different densities of receptor; spontaneous aggregates at 270,000 receptors/cell (open squares), an optimal concentration (10−7 M) of crosslinker at 270,000 receptors/cell (black circles), or optimal crosslinker (10−7 M) at 13,500 receptors/cell (green open circles). The histograms on the right edge of the plot show the remaining number of aggregates at times > 75 msecs. Panel C; 2 simulations, kr (dissociation constant) = 10  or = 100 (green squares) both at 270,000 receptors/cell.Panel A, 3 simulations, all at 270,000 receptors/cell, spontaneous aggregates (open squares), an optimal concentration (10(TIF)Click here for additional data file.