N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB13. This results in a 2-fold increase in kcat/Km for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves kcat/Km by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in kcat/Km, with little apparent change in kcat. A peptide that binds to the DRS of ERK2 affects Km, but not kcat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the  In contrast, the F-site is a smaller hydrophobic motif, ΨXΨP, where Ψ is aromatic chg), containing two common-docking Asp residues and a nearby hydrophobic docking groove (Φhyd) constitute the D-recruitment site (DRS) of ERK2), while the FRS is a relatively shallow hydrophobic pocket found adjacent to the Ser/Thr-Pro binding site. MAPK docking interactions are mediated through MAPK recruitment sites, which are known to recognize modular-docking sequences called docking sites Pointed domain  domain) N-terminus and proposed a model for the interaction between ERK2 and Ets-1 where the SAM domain and the N-terminus interact with the FRS and the DRS of ERK2, respectively  to ERK2. The modeling suggests that the N-terminus remains disordered, with residues 10–16 loosely associating with the hydrophobic groove of the DRS. A putative ΦA-X-ΦB motif is identified comprised of 11LTI13. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction. To our knowledge, this represents the first model for the structure of an activated MAPK•substrate complex.In the present study we use a computational approach complimented by steady-state kinetic experiments to extend our understanding further to elucidate a more detailed model for the binding of residues 1–138 of Ets-1 . Yeast extract, tryptone, agar, and IPTG were obtained from US Biologicals . Ni-NTA agarose, Quiaprep Spin miniprep Kit, PCR QIAquick Purification Kit, and QIAEX II Gel Extraction Kit were supplied by Qiagen Inc. . Restriction enzymes, PCR reagents, and T4 DNA ligase were purchased from New England Biolabs  and Invitrogen Corp. . Oligonucleotides were purchased from Sigma. A Mono Q HR 10/10 anion exchange column was purchased from Amersham Biosciences . The Escherichia coli strain DH5α, used for cloning and mutagenesis, and the strains BL21 (DE3) used for recombinant protein expression, were obtained from Invitrogen. The pET28a vector was purchased from Novagen. The remaining molecular biology reagents, including DNA ladders and protein molecular mass standards, were obtained from Invitrogen Corp. All other buffer components and chemicals were obtained from Sigma.NovaSyn TGR resin was purchased from Novabiochem . Fmoc-6-aminohexanoic acid was purchased from AnaSpec . Other Fmoc-amino acids, HBTU, and HOBT were obtained from Advanced ChemTech . Ultrapure grade Tris and HEPES were obtained from Sigma . MP Biomedicals  supplied [N-terminus and then cleaved using a cleavage cocktail . The cleaved peptides were precipitated with 45 mL ethyl ether. After centrifugation the ethyl ether was removed and the precipitated peptides resuspended in 15% aqueous acetonitrile and freeze-dried. Crude peptides were purified by HPLC using a reverse phase C-18 column  on an AKTA system (Amersham) using a gradient of H20 (0.1% trifluoroacetic acid) against acetonitrile (0.1% trifluoroacetic acid) with a flow rate of 2 mL/min. The peptides were subjected to an elution profile of 0–10% acetonitrile 0–5 min; 10–30% 5–60 min. Purified peptides were characterized for purity and mass by analytical HPLC  followed by mass spectrometry using either a MALDI-TOF  or an ESI . The following analytical results were obtained; Lig-F, observed 1860.01, calculated mass 1860.99; Lig-D, observed 2209.98, calculated mass 2211.31).Peptides were synthesized on rink resin (NovaSyn TGR resin) using a solid phase peptide synthesizer  was modified by PCR using site directed mutagenesis to construct an N-terminal truncation mutant containing Ets-1 residues 24-138 (Δ23N-Ets) with an initial methionine (pET-28a ΔN23Ets).A pET-28a bacterial expression vector encoding a hexa-histidine tag followed by the cDNA encoding murine Ets-1 residues 1-138  followed by the codon encoding Phe-24 (5′-GG GAA TTC CAT ATG TTC CCT TCC CCG GAC ATG-3′) and an outer reverse primer (5′-GCT AGT TAT TGC TCA GCG GTG G-3′) using the following PCR conditions: 94°C for 5 min to denature the complementary strands; 30 cycles of 55°C for 30 sec to anneal the primers, extension for 1 min at 72°C, followed by a denaturation step at 94°C for 45 sec; complementary strands were extended a final 10 min at 72°C. The N-terminal mutant PCR product was digested with NdeI and HindIII and ligated into NdeI-HindIII digested pET-28a. All proteins produced from pET-28a have an N-terminal sequence of M-G-S-S-H-H-H-H-H-H-S-S-G-L-V-P-R-G-S-H- prior to the initial methionine encoded by the Ets cDNA giving EtsΔ24-138 an approximate mass of 15, 391 Da whereas Ets 1-138 has a mass of 17, 681 Da .pET-28a Ets was modified by overlap extension polymerase chain reaction to construct the single cysteine mutant of Ets (C99A/C106A/C112A) in an Ets S26A background as reported elsewhere 2, 3% DMSO, 80 ng of template DNA (Ets-C31 pET28a vector), 0.5 µM primers forward and reverse, and 1 µL of Phusion DNA polymerase . The cycling parameters were 98°C for 2 min, followed by 35 cycles at 98°C for 10 s, 72°C for 30 s, with a final elongation step 72°C for 10 min. Forward primers  (NheI site and mutations are in bold) and reverse primer (5-CAG CAA GCT TGCAAA GAG GAT GTG AAA TAA  -3) (HindIII site in bold) were used to generate the mutants. The PCR product was digested with NheI and HindIII, ligated into the NheI-HindIII digested pET28a vector and then transformed into DH5α E. coli cells. The construct was verified by sequencing the DNA at the UT core facilitiespET28a-Activated tagless ERK2 was generated essentially as described in . Expression and purification of Ets-1 (1-138) was followed by the method described in the previously published literature 2) containing 2 nM ERK2 and varied concentrations of substrates and inhibitors. Rates were measured under conditions where total product formation represented less than 10% of the initial substrate concentrations. The reaction was incubated for 10 min before initiation by addition of enzyme and quantified as described previously v against 1/s were checked for linearity, before the data were fitted to eqn. 1 using a non-linear least squares approach, assuming equal variance for velocities, using the program Kaleidagraph 3.5 (Synergy software). The intercepts i) (for inhibition experiments) or the reciprocal of the non-varied substrate concentration (1/s) . These plots were used to determine the appearance of the overall kinetic equation. Values for kinetic constants were then obtained using the program Scientist (Micromath) by fitting the kinetic data to the relevant over-all equation. Data conforming to a sequential initial velocity pattern were fitted to eqn 2; data conforming to linear competitive inhibition were fitted to eqn. 3; data conforming to hyperbolic mixed inhibition were fitted to eqn. 4. Dose-response curves for data conforming to linear inhibition were fitted to eqn. 5; and for data conforming to hyperbolic inhibition to eqn. 6.zReactions were carried out at 27°C in kinase assay buffer , coordinates were first obtained for the fragment RRNLKGLNLNL using the known structure of this fragment bound to the MAPK FUS3 (PDB ID: 2B9H) N-terminus using homology software (Modeller9v4) For sequence YAPRAPAKLAFQFPSR (Lig-F) - coordinates were first obtained for the FSFG motif from mRNA export factor MEX67 (PDB ID: 2KHH) Residues 1-28 of Ets-1 were modeled onto Ets-1 (residue 29-138) (PDB ID: 2JV3) RRGSNVALMLDC) derived from the protein phosphatase HePTP in complex with inactive ERK2 (PDBID: 2GPH) RRNLKGLNLNL (Lig-D) and ERK2 were set according to the known interactions of the two motifs (RR and ΦA-X-ΦB) with the phosphatase. The structure of 2GPH indicates that ΦB in the ΦA-X-ΦB motif will make Van der Waals contacts with Leu-113 of αD in ERK2, Phe-127 of αE, Leu-155 of β7, Cys-159 in the β7-β8 loop, and Thr-108 in the crossover connection. ΦA binds to a hydrophobic patch on the surface of helix αE in ERK2, formed by His-123 and Tyr-126, and Cys-159 in the β7-β8 hairpin. Asp-316 and Asp-319 in the CD site of ERK2 can form direct contacts with the RR motif FQFPSR). These interactions were also utilized as loose constraints in the docking.a) Binding within the DRS – The crystal structure of a docking site peptide  1-42 docking to ERK2 were predicted using GOLD 4.1 ). Following the strategy previously adopted to build Model A, the Thr-38/Pro-39 motif of Ets was restricted to the active site of ERK2 N-terminus adopts a disordered conformation, with a stretch of seven residues, 10TLTIIKT16, loosely engaging the hydrophobic groove of the DRS of ERK2  Ets complex, it is nevertheless of significant value, because it provides an important framework from which to begin to understand the specificity and mechanism of ERK2 towards Ets-1 and potentially towards other substrates also. Significantly, when we examine which amino acids undergo perturbations in their NMR chemical shifts in inactive ERK2 in the presence of an excess of Ets we see good agreement with our model , His-123 (0.14 ppm), Tyr-126 (0.1 ppm) and Cys-159 (0.21 ppm). Additionally at the FRS, for residues where chemical shift assignments are available, large perturbations are seen for Asn-260 (0.17 ppm) and Leu-262 (0.14 ppm) on a2L14 helix of the MAPK insert. This suggests that the topology of the model is accurate with Ets engaging both the DRS and the FRS in both inactive and activated ERK2. A more comprehensive listing of residues that undergo significant chemical shift changes is shown in submitted).The resulting model of the ERK2•ur model . Indeed,ur model  are seenEts and the FRS of ERK2 we designed a modular peptide ligand for the hydrophobic binding pocket of the FRS formed by the P+1 site, the αF helix, and the MAP kinase insert N-terminus of Lig-F has conformational flexibility and can adopt a number of binding modes with the surface of ERK2.To assess the importance of the interaction between the SAM domain of Ets at several fixed concentrations of Lig-F was assessed. When the kinetic data were plotted in double reciprocal form a mechanism of competitive inhibition was revealed  of Ets) is plotted against the concentration of Lig-D (0–400 µM)  binds to the DRS of ERK2. A notable feature of the N-terminus of Ets-1 is that while it does not contain such a consensus sequence residues 10-16 contain a potential ΦA-X-ΦB motif. Interestingly, the modeling placed Leu-11 and Ile-13 in the Φhyd subsite in the proximity of the Ø1 and Ø2 pockets respectively, suggesting that this motif may contribute to the association of the N-terminus of Ets-1 with ERK2. To further examine the model several mutations were introduced into the N-terminus of Ets-1 that were predicted to destabilize the interaction of the putative 11ΦA-X-ΦB13 motif. Specifically, the following mutants of Ets, Asp11, Asp13, Ala13/Ala14 and Asp13/Asp14 were prepared. The introduction of these mutations resulted in small yet significant effects on the steady-state kinetic parameters that are generally consistent with the notion that Leu-11 and Ile-13 (or Ile-14) interacts loosely with the DRS of ERK2 to promote binding 2-ΦA-X-ΦB motif with potential to interact at the Φchg site. This mutant exhibits a 1.7-fold improved specificity (as determined by comparing kcat/Km) over Ets, which is largely due to the 3-fold decrease in Km. While the introduction of a second Arg at residue position 6 had no additional effect it was interesting to note that when the N-terminus was exchanged for the corresponding docking sites of Elk-1 and MKK1 (which conform to R/K-(X)5-ΦA-X-ΦB and R/K-(X)3-ΦA-X-ΦB consensus sequence respectively) the resulting proteins were also better substrate than Ets by a factor of approximately 2-fold. Taken together, the mutagenesis studies support the notion that the N-terminus of Ets serves as a weak ligand for the Φhyd site of the DRS of ERK2. A conclusion supported by the NMR studies . It is intriguing that despite the loss of the N-terminal interaction, kcat for the phosphorylation of Δ23N-Ets is almost identical to that for the phosphorylation of Ets, suggesting that the tether does not significantly influence steps associated with turnover once the complex is formed. Consistent with this is the observation that mutations in the DRS have little influence on kcat, see Table 2 in Abramczyk et al. .A careful analysis of the kinetic mechanism of inhibition by Lig-D reveals that it inhibits hibition . A reasoΔ23N-Ets . FurtherN-terminus of Ets-1 is that while it does not contain a conventional consensus sequence for binding to the DRS of ERK2 it contains a potential ΦA-X-ΦB motif. Interestingly, 4 out of the top 5 structures from 5 independent virtual docking runs suggested that Ile-13 of Ets-1 binds the Ø2 site of the DRS -X2-ΦA-X-ΦB consensus sequence into the N-terminus we were able to slightly increase the specificity. Thus, ERK2 exhibits a 7-fold higher specificity for Ets L7R over Ets L11D. ERK2 is reported to phosphorylate Ets-2, which is related in sequence to Ets-1 N-terminus of Ets-1 possesses little sequence similarity to the corresponding region of Ets-2 A notable feature of the  the DRS . RecentlN-terminus of Ets-1 remains essentially disordered upon binding to ERK2 and engages the DRS of ERK2. While the model shows Thr-38 bound to the active site we believe that given the disordered nature of the loop it may adopt several alternative conformations that are similar in energy. A binding study on a series of Thr-Pro mutants would appear to support the notion that the binding of the Thr-Pro motif does not contribute to the stability of the ERK2•Ets complex The modeling studies described here suggest that the C-terminus of the protein kinase MK2 contains a docking site for the DRS of its activator p38MAPKα Ets to ERK2 and found that it binds with a rate that exceeds the rate of diffusion for the association of two proteins, with a rate constant for association of kon  = 5×106 M-1s-1 and with rate constant for dissociation of k >100 s-1Since the seminal paper by Wright Ets complex suggests that Ets-1 simultaneously binds both the DRS and the FRS of ERK2 to position the phosphorylation site, Thr-38, in the proximity of the active site. Previous stopped-flow studies, which suggest that the association between ERK2 and Ets is both rapid and transient, raise intriguing questions about the role of the two docking sites. As cell signaling is a dynamic process, where proteins at low concentrations must often interact and dissociate rapidly and yet bind with high specificity, highly evolved protein-protein interactions are expected, to facilitate efficient recognition. The new model allows us to begin to examine the mechanism of binding as well as the conformational dynamics associated with phosphorylation once the enzyme substrate complex is formed. With further NMR studies on the horizon we hope to further refine the structure using a combination of computational and experimental approaches.The structural model of the ERK2•Table S1A-X-ΦB) with phosphatase (PDB ID: 2GPH).Distance constraints between Lig-D and ERK2 were set based on the known interactions of the two motifs (RR and Φ Distance constraints between Lig-F and ERK2 were set to make the Phe-Xaa-Phe motif (DEF motif) binding a hydrophobic pocket formed between the P+1 site, the αF helix and the MAP kinase insert. The binding constraints for Ets(1-42) were imposed to enforce the hydrogen bonding interactions between Thr residue in the Thr-Pro motif with Asp-147 and Lys-149 of ERK2. In addition, the proline in the Thr-Pro motif was restrained to adopt a similar binding mode to that in Ser-Pro motif of HHASPRK bound to the cyclin-dependent kinase (CDK2) (PDB ID: 1QMZ).(DOCX)Click here for additional data file.


We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.


Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways.In gastric cancer cell line AGS, expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA (Ad-PKG II) and treating the cells with cGMP analogue 8-pCPT-cGMP. Phosphorylation of proteins was detected by Western Blotting and active small G protein Ras and Rac1 was measured by “Pull-down” method. Cell migration activity was detected with trans-well equipment. Binding between PKG II and EGFR was detected with Co-IP. The results showed EGF stimulated migration of AGS cell and the effect was related to PLCγ1 and ERK-mediated signal transduction pathways. PKG II inhibited EGF-induced migration activity and blocked EGF-initiated signal transduction of PLCγ1 and MAPK/ERK-mediated pathways through preventing EGF-induced Tyr 992 and Tyr 1068 phosphorylation of EGFR. PKG II bound with EGFR and caused threonine phosphorylation of it.Our results systemically confirms the inhibition of PKG II on EGF-induced migration and related signal transduction of PLCγ1 and MAPK/ERK-mediated pathways, indicating that PKG II has a fargoing inhibition on EGF/EGFR related signal transduction and biological activities of gastric cancer cells through phosphorylating EGFR and blocking the activation of it. In 2004, Cook Cell migration is important in normal physiology and in disease. Acquisition of migratory ability by cancer cells is a characteristic that contributes to spread of metastatic tumor cells to distant organs. Recent data showed that EGF could stimulate the migration of cancer cells. The mechanism through which EGF stimulates cell migration is not clear but some data indicated that EGF may do this through initiating signal transduction of PLCγ1 and MAPK/ERK-mediated pathways. In this experiment, we detected the migration-stimulating effect of EGF in AGS cells and used inhibitor of key components in the signal pathways to investigate the possible signal transduction associated with the effect. The results showed that EGF treatment increased the migration activity of AGS cells and both MEK (key component of MAPK/ERK-mediated pathway) inhibitor U0126 and PLCγ1 inhibitor U73122 inhibited EGF-induced migration, indicating that EGF stimulated cell migration activity through activating both MAPK/ERK and PLCγ1 mediated signal transduction pathways .When EGF binds with EGFR, it causes auto-phosphorylation of the receptor. There are several auto-phosphorylation sites which are connected to different signal transduction pathway. Tyrosine 992 and Tyrosine 1068 are among the auto-phosphorylation sites of EGFR and are associated with PLCγ1-mediated and MAPK/ERK-mediated signaling respectively. In this experiment, we investigated the inhibitory effect of PKG II on the Tyrosine 992 and Tyrosine 1068 phosphorylation of EGFR in differently treated AGS cells by using Western blotting. The results showed that EGF treatment caused a 14 folds increase of Tyrosine 992 and an 8 folds increase of Tyrosine 1068 phosphorylation of EGFR. In cells infected with Ad-PKG II and stimulated with cGMP, the phosphorylation was significantly decreased , 3. ThisPLCγ is the downstream component of receptor tyrosine kinases (RTKs). It has two isoforms: PLCγ1 is ubiquitously distributed and PLCγ2 is expressed mainly in hematopoietic cells. Activation of PLCγ1 requires its recruitment to the membrane and association, through its SH2 domain, with activated RTKs such as EGFR. This association will result in the phosphorylation of PLCγ1 on tyrosine residues, particularly on tyrosine 783, and an increase of its enzymatic activity. We applied IP method to isolate PLCγ1 and then used Western blot method to detect the phosphorylation of PLCγ1. The results showed that EGF treatment caused an obvious increase of Tyr783 phosphorylation of PLCγ1 and the increase of PKG II activity through infecting the cells with Ad-PKG II and stimulating the cells with cGMP efficiently prevented the EGF-induced phosphorylation of PLCγ1 .2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), two molecules that regulate the mobilization of intracellular Ca2+ and protein kinase C activity respectively. To observe the effects of EGF and PKG II on the formation of DAG, ELISA method was used to detect DAG concentration in AGS cells. The results showed that in EGF stimulated AGS cells, the level of DAG increased obviously and pre-infection with Ad-PKG II and treatment with 8p-CPT-cGMP inhibited the formation of DAG caused by stimulation with EGF  to cytoplasm and high expression and activity of PKG II significantly inhibited the release, reversing the effect of EGF treatment -dependent kinase II alpha (CaMKIIα), which is a primary isoform of CaMKII, have suggested that when Ca2+/CaM binds with CaMKIIα, intra-molecular autophosphorylation occurs and the phosphorylation maintains the persistent activation of the enzyme. The antibody against phospho-CaMKIIα (Thr286) was applied in Western blotting to detect the phosphorylation of CaMKIIα. The results showed that in AGS cell treated with EGF, Thr286 phosphorylation of CaMKIIα increased by nearly 2.5 folds and infection with Ad-PKG II and treatment with 8-pCPT-cGMP inhibited the increase of the phosphorylation induced by EGF (Studies on the regulation of Cad by EGF .MAPK/ERK is the key component of MAPK/ERK-mediated pathway. Phosphorylation at both threonine 202 and tyrosine 204 residues of ERK1 and threonine 185 and tyrosine 187 residues of ERK2 is required for full enzymatic activation. The antibody against p-ERK1/2 (Thr 202/Tyr 204) was applied in Western blotting to detect the dual phosphorylation of ERK. The results showed that within five minutes after adding EGF to cell culture medium, phosphorylation of ERK1/2 in AGS cells increased dramatically (10 folds) and the phosphorylation was inhibited by pre-infecting the cells with Ad-PKG II and activating the enzyme with 8-pCPT-cGMP .Small G protein Ras is another key component in MAPK/ERK-mediated signal pathway. It has two forms in cells: GTP-bound active form and GDP-bound inactive form. Once Ras is in GTP-bound form, it can bind and activate Raf-1 and start the consequent activations of serine/threonine kinases in the signal pathway. We applied “pull-down” method to detect the activated Ras. The result showed that after adding EGF to the culture medium, active Ras in AGS cells increased obviously within five minutes. Infecting the cells with Ad-PKG II and stimulating them with 8-pCPT-cGMP before adding EGF significantly prevented the EGF-induced Ras activation .Small G protein RAC1 is the main member of Rho family which play important role in regulating migration of cancer cells. Both PLCγ1 and MAPK/ERK mediated signal transduction can activate RAC1 and thereafter stimulate cell migration. To further confirm the inhibitory effect of PKG II on EGF/EGFR-induced signaling which is related to migration, “Pull-down” method was applied to detect the inhibition of PKG II on activation of RAC1. The result showed that EGF treatment caused an obvious increase of active RAC1 and high activity of PKG II efficiently inhibited the activation of RAC1 . This prThe mechanism through which PKGII blocks the EGF-induced activation of EGFR was preliminarily investigated in this experiment. Co-Immunoprecipitation was applied to detect the interaction between PKGII and EGFR. Western blotting with pan anti phosphorylation of threonine antibody was used to detect the Threonine phosphorylation of EGFR caused by PKGII. The results of Co-Immunoprecipitation showed that in AGS cells infected with Ad-PKG II and stimulated with 8-CPT-cGMP, direct binding between PKG II and EGFR was detected , panel ACurrently, two cGMP-dependent protein kinases (PKGs), PKG I and PKG II, have been identified in mammalian cells EGFR exists on the surface of all cells. With a molecular weight of 170KD, EGFR has an extracellular domain, a cross-membrane domain and an intracellular domain. The intracellular domain of EGFR has 542 amino-acid residues and can be divided into approximate membrane sub-domain, tyrosine kinase sub-domain, and C-terminal sub-domain In this paper, we investigated the action of PKG II on EGF-induced migration activity of gastric cancer cell line AGS. The result showed that PKG II had significant inhibition on cell migration caused by EGF. This provides further evidence for revealing the tumor inhibitory effect of PKG II. Research data have shown that among the EGF/EGFR initiated signal transduction pathways, PLCγ1 and MAPK/ERK mediated signal transduction pathways are related to migration activity The signal transduction of both PLCγ1 and MAP/ERK-mediated pathways can activate small G protein Rac1, which is a key component in regulating cell migration In vitro experiments confirmed that over-expression of EGFR caused transformation of NIH-3T3, Rat-1 and NRK cells and blocking EGFR activation inhibited proliferation of some tumor cells EGFR is closely associated with tumorigenensis. Over expression and mutation of EGFR often happen in most cancers. Research data showed that over 50%–70% of lung cancer, colon cancer and breast cancer have high expression of EGFR Research data showed that some protein kinases can cause phosphorylation of EGFR and affect its activation and/or destination. For example, protein kinase C (PKC) can cause the phosphorylation of Threonine 654 of EGFR and regulates receptor binding and internalization 2+ indicator Fluo-3/AM and Membrane and Cytosol Protein Extraction Kit were from Beyotime . Human diacyl glycerol (DAG/DG) ELISA Kit was from Cusabio Biotech Co., Ltd. . All other reagents used were of analytical grade.Human gastric cancer cell line AGS was provided by the Institute of Cell Biology . Adenoviral vectors encoding β-galactosidase (pAd-LacZ) and PKG II (pAd-PKG II) were kind gifts from Dr. Gerry Boss and Dr. Renate Pilz in University of California, San Diego, U.S.A. Dulbecco’s Modified Eagle’s Media (DMEM) and fetal bovine serum (FBS) were from GIBCO . The antibody against PKG II was from ABGENT Biotechnology . Goat anti-β-actin, mouse anti-pan-Ras, and mouse anti-PLCγ1 antibodies were from Santa Cruz . Rabbit anti-p-EGFR (Tyr1068), rabbit anti-p-EGFR (Tyr992), rabbit anti-EGFR, mouse anti-p-ERK(Thr 202/Tyr 204), rabbit anti-ERK and rabbit anti-p-PLCγ1(Tyr783) antibodies were from Cell Signaling Technology . Rabbit anti-PKCα and rabbit anti-p-CaMK IIα (Thr286) were from Bioworld Technology . Rabbit anti-p-Thr antibody was from Abcam .Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson Immuno Research Laboratories . The cellular permeable cGMP analog 8-pCPT-cGMP was from Calbiochem . EGF, U73122 and U0126 were from Sigma . Electrochemiluminescence (ECL) reagents were from Millipore . Ca293A cells were transfected with adenoviral vector encoding LacZ and PKG II respectively and cultured for up to ten days until CPE was seen. The cells and the culture medium were harvested and underwent three freezing-thawing cycles. The supernatant containing adenoviruses (Ad-LacZ and Ad-PKG II ) were used to infect new 293A cells to amplify adenoviruses. The amplified adenoviral preparations were titrated and the pfu/ml was determined, and kept in −80°C until use.2. The medium was changed every two days and the cells were sub-cultured at confluence. On the day before infection, cells were freshly planted at 70–80% confluence, and the infection with Ad-LacZ and Ad-PKG II was performed.AGS cells were cultured in DMEM supplied with 10% FBS and maintained at 37°C in a humidified incubator with 95% air and 5% COProtein samples were subjected to SDS-PAGE (8–12%) gel according to the molecular size of target protein, and electrophoresis and membrane transfer was performed following the manufacturer’s protocol . The primary antibodies were incubated over night at 4°C in TBS-T (2% Tween-20), and the corresponding secondary antibodies were incubated for 1 h at RT in TBS-T (2% Tween-20), with three washes after each incubation. ECL reagents were used to show the positive bands on the membrane. To perform densitometry analysis, digital images of the positive bands were obtained with Chemidoc XRS and analyzed using the image analysis program Quantity One . The results were showed as the ratio of target protein/loading control.2, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The sample was collected and centrifuged  to get rid of the debris. The supernatant was incubated with glutathione-Sepharose beads and glutathione S-transferase-Ras-RBD (GST-RBD) at 4°C for 1 h. The beads were washed 3 times with lysis buffer and heated in boiled water to release proteins. The protein samples (containing active Ras) were analyzed by Western Blotting with antibody against pan-Ras. The active Rac1 was detected with similar method but with GST-Pak1 protein binding domain (GST-PBD) and antibody against Rac1.The activity of Ras was detected with Pull-down method as described previously 3VO4) per plate. Antibodies against PLCγ1 and p-PLCγ1 were used for immunoprecipitation. The precipitates were probed with antibodies against target proteins.The cells growing on 100-mm culture plate were washed two times with cold PBS and lysed by adding 1 ml RIPA buffer  ester for 30 min at 37°C in DMEM. After the loading, cells were washed with PBS and suspended in DMEM. Fluorescence measurements were performed using an Olympus Fluoview-500 confocal system. Fluo-3 was excited by argon laser light at 488 nm and fluorescence was measured at wavelengths of 515 nm.DAG concentrations were measured by ELISA, according to the manufacturer’s instruction. This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for DAG has been pre-coated onto a micro-plate. Standards and samples are pipetted into the wells and any DAG present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for DAG is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of DAG bound in the initial step. The color development is stopped and the intensity of the color is measured.Subcellular fractionation into cytosol and membrane fractions was performed by using Membrane and Cytosol Protein Extraction Kit, according to the manufacturer’s instruction. Monolayer cultures were washed three times with ice-cold PBS solution and scraped into cold homogenization buffer (HB) containing 20 mM Tris–HCl (pH 7.4), 4 mM EDTA, 2 mM EGTA, 10% glycerol, 10 g/ml leupeptin, and 1 mM PMSF. The cells were lysed via sonication with 4–15 s intervals and complete lysis was monitored microscopically. The homogenate was ultracentrifuged at 86,000 g for 45 min at 4°C, and the supernatant was designated as the cytosolic fraction. The pellet was resuspended in HB containing 1% Triton X-100 and incubated on ice for 30 min. The samples were then centrifuged at 14,000 g for 20 min at 4°C, and the supernatant was designated as the membrane fraction. All samples were boiled and cleared by centrifugation.4cells were seeded into the upper chamber containing culture medium without FBS. Cell migration to the bottom side of membrane was induced by medium containing 10% FBS in the lower chamber for 12 h at 37°C in a tissue culture incubator. Migrated cells on the bottom side of the membrane were fixed in 40% paraformaldehyde solution for 30 min, stained in Giemsa solution for 10 min, and then rinsed in water. The stained cells were subjected to microscopic examination under a light microscope. Migrated cells were counted in 5 randomly selected fields per insert, and the values were averaged. All experiments were performed with three replicates under each migration condition.Migration activity of AGS cells were detected by transwell system . After trypsinization, 5*10P-value of less than 0.05 was considered significant.The data were expressed as means ± standard deviation (SD). Statistical analysis was performed using a two-tailed ANOVA with SPSS statistical software. A


Salt marsh die-off is a Western Atlantic conservation problem that has recently spread into Narragansett Bay, Rhode Island, USA. It has been hypothesized to be driven by: 1) eutrophication decreasing plant investment into belowground biomass causing plant collapse, 2) boat wakes eroding creek banks, 3) pollution or disease affecting plant health, 4) substrate hardness controlling herbivorous crab distributions and 5) trophic dysfunction releasing herbivorous crabs from predator control. To distinguish between these hypotheses we quantified these variables at 14 Narragansett Bay salt marshes where die-off intensity ranged from <5% to nearly 98%. Nitrogen availability, wave intensity and plant growth did not explain any variation in die-off. Herbivory explained 73% of inter-site variation in die-off and predator control of herbivores and substrate hardness also varied significantly with die-off. This suggests that salt marsh die-off is being largely driven by intense herbivory via the release of herbivorous crabs from predator control. Our results and those from other marsh systems suggest that consumer control may not simply be a factor to consider in marsh conservation, but with widespread predator depletion impacting near shore habitats globally, trophic dysfunction and runaway consumption may be the largest and most urgent management challenge for salt marsh conservation. Spartina alterniflora are occurring in New England, driven by predator depletion and release of the herbivorous marsh crab Sesarma reticulatum (hereafter Sesarma) from consumer control Salt marsh die-off is a growing phenomenon in the western Atlantic While it has been hypothesized that human impacts causing trophic dysfunction are driving these die-offs, salt marshes are widely considered to be controlled exclusively by physical forces Recently, creek bank salt marsh die-off has spread into Narragansett Bay, where it had been reported absent as recently as 2009 All necessary permits for field sampling were obtained from the Cape Cod National Seashore.Fourteen accessible salt marshes with a range of die-off conditions were identified in Narragansett Bay. The extent of die-off was measured at each site in July 2013 by walking two randomly placed 100 m creek bank transects and every 10 m measuring the total width of the cordgrass zone and the width of the cordgrass zone denuded by die-off.We quantified the history of die-off at each site using archived aerial photographs from 1997, 2003, 2008, and 2012. Marsh habitat was defined as the total area covered in visible salt marsh vegetation and die-off area was defined as the total area converted from marsh vegetation to barren peat over each time interval. All areas were defined and quantified using ArcGIS. Marsh linear extent was defined as the total length of marsh edge bordering water (length of marsh border exposed to die-off conditions). To test the hypothesis that salt marsh die-off in Narragansett Bay is recent, percent linear extent affected by die-off was calculated for each site and time point. We tested the hypothesis that the intensity of die-off is increasing over time by measuring the mean width of die-off and the mean rate of die-off expansion for each site and time period. Mean die-off width was calculated as total area of die-off divided by length of border affected. Rate of die-off expansion was calculated as mean die-off width per year. An ANOVA with a univariate split plot approach was used to analyze both percent linear extent and the width of die-off over time.Spartina alterniflora (hereafter cordgrass) was transplanted to the creek bank of each site. It was protected from crab, bird and mammal herbivory by a 1 cm mesh hardware cloth top-less cylindrical cage that extended 20 cm belowground and 30 cm aboveground, a height tall enough to exclude any herbivores. Mesh size was chosen to allow ample sunlight and room for cordgrass growth. Cage controls were not necessary since extensive previous work on Cape Cod and Long Island Sound showed no caging effects on growth using this technique In May 2013, an undamaged common source core (7 cm diameter) of short-form To examine the hypothesis that eutrophication contributes to inter-site variation in creek bank die-off To examine the hypothesis that boat wakes or wave exposure contributed to creek bank die-off via erosion Sesarma herbivory was measured at each site the second week of July 2013. Herbivory was estimated by walking a 20 m transect along the creek bank of each site and every 2 m scoring 100 cordgrass stems for crab herbivore damage in a 50×50 cm quadrat. Sesarma grazing leaves characteristic rasped edges and clipped blades on transplanted cordgrass Substrate hardness, an accurate proxy for peat density Sesarma leading to increased Sesarma densities and herbivory, we quantified Sesarma densities, predation pressure on tethered Sesarma, and top predator biomass at each study site.To quantify trophic dysfunction in Narragansett Bay, operationally defined as how severely predator depletion has reduced predation pressure on Sesarma abundance was estimated with nocturnal pitfall traps in late July when the crabs showed maximum activity (n  =  8 traps/site) Sesarma were counted and measured the following morning. Sesarma tethering assessed inter-site variation in predation pressure. Sesarma were tethered to braided monofilament fishing line, secured with cyanoacrylic glue, and fastened using metal pins in Sesarma habitat at each site (n  =  15 Sesarma/site). Caged crabs to assess tethering artifacts were not deployed because previous studies on Cape Cod and Long Island Sound found no tethering artifacts Sesarma behave normally on tethers, digging shallow burrows and foraging in the tethered area. Crabs were deployed overnight in August 2013 and mortality was scored the following morning. Predation mortality left broken carapace fragments attached to tethers, mortality from desiccation left dead intact crabs, while surviving tethered crabs were found in newly excavated shallow burrows.Sesarma predators in the past and thus provide reliable estimates of predatory populations Inter-site variation in predator populations was quantified with baited, commercial 30×60×90 cm traps with funnel openings (n  =  3 traps/site) in August 2013. Traps were constructed of 2.5 cm PVC-coated steel mesh and deployed overnight in creeks >100 m apart. Traps have been shown to catch dominant SesarmaSesarma burrowing and herbivory, we also performed linear regressions between predator biomass, predation rates on Sesarma, substrate hardness and herbivory. All variables were transformed as needed when necessary. Data was analyzed in JMP 10.Multiple regression with leaf tissue percent nitrogen, cordgrass transplant biomass, chalk block dissolution and herbivore intensity was used to examine alternative hypotheses explaining inter-site variation die-off among sites. To examine the hypotheses that site differences in herbivory were being driven by inter-site differences in top predator abundance and predation on 2,12  =  394.02, P < 0.0001). Die-off was absent in Narragansett Bay in 1997, minimal (7%) in 2003, but by 2012 affected >85% of creek bank habitats  nearly 3-fold between 2008 and 2012  significantly increased over time  and herbivory to test the hypothesis that substrate hardness dictates crab herbivory. Variation in substrate hardness in vegetated areas along the grazing border explained 46% of variation in salt marsh creek bank die-off (F1,12  =  12.55, P < 0.01, Sesarma abundance, explaining 39% of inter-site variation in Sesarma abundance (F1,12  =  7.81, P < 0.05, Sesarma abundance was positively correlated with herbivory, explaining 30% of the inter-site variation in herbivory across sites (F1,12  =  5.30, P < 0.05, To test the hypothesis that creek bank die-off is caused by trophic dysfunction due to overfishing, we performed regressions across trophic levels  to test Our results reveal that the spread of creek bank die-off into Narragansett Bay salt marshes is being driven by predator depletion, which releases herbivorous crabs from consumer control. Additionally, salt marshes with medium substrate hardness are the most vulnerable to die-off. Our results reject the alternate hypotheses that eutrophication, boat wakes, plant disease and/or pollution are associated with the spread of salt marsh die-off into Narragansett Bay and suggest that they are not drivers of the widespread die-off of western Atlantic salt marshes.Sesarma burrowing which increases vulnerability to calving and marsh loss While many explanations have been proposed for salt marsh die-off, few have been rigorously tested by well-replicated field experiments. We found no evidence that eutrophication , pollutiOf all of the hypotheses proposed to cause die-off, herbivory remains the only one that has been widely examined experimentally in well-replicated studies across the Canadian subarctic Historically, salt marsh ecosystems have been thought to be controlled by physical forces based on correlations between marsh productivity and physical factors Our results predict a dire future for New England salt marshes. Salt marshes are one of the most valuable ecosystems on earth per unit area because they provide coastal storm buffering, biochemical processing, carbon sequestration, and nursery habitat ecosystem services


The structure of a new crystal form of IgE-Fc in complex with its B-cell receptor CD23 has been determined. The structure reveals that there is conformational variability at the interface in both IgE-Fc and CD23. The antibody IgE plays a central role in allergic disease, functioning principally through two cell-surface receptors: Fc∊RI and CD23. Fc∊RI on mast cells and basophils mediates the immediate hypersensitivity response, whilst the interaction of IgE with CD23 on B cells regulates IgE production. Crystal structures of the lectin-like ‘head’ domain of CD23 alone and bound to a subfragment of IgE consisting of the dimer of C∊3 and C∊4 domains (Fc∊3-4) have recently been determined, revealing flexibility in the IgE-binding site of CD23. Here, a new crystal form of the CD23–Fc∊3-4 complex with different molecular-packing constraints is reported, which together with the earlier results demonstrates that conformational variability at the interface extends additionally to the IgE Fc and the quaternary structure of its domains. Fc∊RI binds IgE with high affinity (Ka = 1010–1011 M−1) and is responsible for allergic sensitization and the immediate hypersensitivity response in which minute amounts of allergen cross-link Fc∊RI-bound IgE on mast cells and basophils, triggering cellular degranulation. The IgE-binding α-chain of Fc∊RI consists of two extracellular Ig-like domains  superfamily  and eluted with 25 mM Tris–HCl pH 7.5, 200 mM NaCl. Pooled fractions were concentrated to 1 ml and loaded onto a HiLoad 16/60 Superdex G75 column  pre-equilibrated and subsequently washed with purification buffer. The correct folding of derCD23 was assessed by one-dimensional 1H-NMR at 500 MHz .Recombinant human derCD23 (Ser156–Glu298) was expressed in et al., 2012M sodium acetate buffer pH 6.0, followed by gel filtration on a Superdex S200 matrix  in PBS pH 7.4.The recombinant human Fc∊3-4 gene  was synthesized, cloned and transiently transfected into HEK293 cells as described previously  sodium azide (storage buffer). derCD23–Fc∊3-4 complex crystals were obtained by the sitting-drop vapour-diffusion method. Crystals were grown by mixing 150 nl of a protein solution consisting of 0.2 mM derCD23 (3.2 mg ml−1), 0.1 mM Fc∊3–4 (5 mg ml−1) with 75 nl reservoir solution consisting of 100 µl 22–26%(w/v) PEG 1500, 20%(v/v) glycerol. The largest crystals, which grew within a week at 291 K in the absence of any added CaCl2, were ∼500 µm in length and were obtained by micro-seeding. Several small plate-like crystals were added to 50 µl reservoir solution, vortexed vigorously for 2 min, diluted 1000-fold with storage buffer and added to the protein solution at a tenth of the final volume before setting up crystallization optimization trials.Prior to crystallization, Fc∊3-4 was concentrated to 13 mg mlDiffraction data were collected at 100 K on beamline I04 of the Diamond Light Source synchrotron, Harwell, England. No additional cryoprotectant solution was required.2.3.MOSFLM , reflections were selected for the Hingefind .3.3.1.et al., 2012P212121) with three complexes in the asymmetric unit, whereas the structure presented here crystallized in a triclinic lattice (P1) with two complexes in the asymmetric unit . This identity extends to the two highly mobile loop regions of derCD23, loop 1 (Leu226–Glu231) and loop 4 (Arg253–Glu257) , a change that leads to further contact with IgE and explains the 30-fold enhanced affinity . In the presence of Ca2+ this range (for the six chains) decreases to 3° , but remarkably in the triclinic form reported here there is virtually no variation at all between the four independent chains , which display an interdomain angle midway between the two extremes. This is illustrated in Fig. 2d).Comparison between all of the CD23-bound Fc∊3-4 structures shows a more restricted but nevertheless still substantial range of interdomain angles. In the Caes Fig. 2b. In th3° Fig. 2c, but rns Fig. 2a, whichC  has 18 contacts, whereas chain D  has 50 C∊3 crystal contacts.  crystal contacts, ranging from 50 to 75 over the four chains, whereas in the orthorhombic crystals a lower number of C∊3 contacts ranging from 18 to 50 is found. Although there is a smaller number of contacts, it is notable that chain et al., 2013et al., 2002The fact that a range of C∊3–C∊4 interdomain angles is compatible with CD23 binding demonstrates that the plasticity seen at the IgE binding site in CD23 (Dhaliwal 3.3.et al., 20122+-bound derCD23–Fc∊3-4 complex, where it forms one of six additional interface interactions as a result of the local structural rearrangement caused by binding of the cation (Yuan et al., 20132+, a further example of the structural variation that occurs at this interface.The derCD23–Fc∊3-4 interface Fig. 3 is dominThese results refine our understanding of the binding of IgE to CD23 and show that there is a remarkable degree of flexibility at the CD23–IgE interface not only in CD23 as reported earlier, but also in IgE. The different sets of crystal-packing forces acting on the complexes in the triclinic and orthorhombic forms, and the different C∊3–C∊4 interdomain angles in the four and six independent views of the interface in the two crystal forms, respectively, provide snapshots of the range of conformations that are compatible with CD23 binding. Understanding the extent of this plasticity may prove to be important in the design of either direct steric blocking agents or allosteric inhibitors of a protein–protein interaction that plays such a key role in the development of allergic disease.CD23–Fc∊3-4 complex, 4ki1PDB reference: 10.1107/S2053230X14003355/tb5065sup1.pdfSupporting Information.. DOI:


Cystatin C is a constitutively expressed and abundant cysteine protease inhibitor within the cerebrospinal fluid (CSF). Recent studies have reported a significant reduction in cystatin C concentration in the CSF of patients with amyotrophic lateral sclerosis (ALS) and several other neurodegenerative diseases, relative to healthy controls. Cystatin C can exhibit both neuroprotective and neurotoxic properties, suggesting that altered CSF cystatin C concentrations could potentially impact the pathogenesis or progression of these disorders. However, it is unclear if alterations in cystatin C concentration result in physiologically relevant differences in its functional activity within the CSF. Measurements of the cysteine protease inhibitory activity of cystatin C within the CSF have not been reported, and the relationship between CSF cystatin C concentration and activity levels in different disease contexts has not been investigated.We used a papain inhibition assay to evaluate the total cystatin C activity in CSF samples from 23 ALS patients, 23 healthy controls, and 23 neurological disease controls. Cystatin C concentrations in these samples were previously measured by ELISA. Correlations between cystatin C concentration and activity were assessed with nonparametric statistics. Activity ratios were compared among diagnostic groups using both one-way ANOVA and repeated measures statistics.Total cystatin C activity was found to be directly proportional to its protein concentration in all subjects, and cystatin C activity was not altered in ALS patients. In addition, our data suggest that cystatin C is the predominant cysteine protease inhibitor in human CSF.Our data demonstrate the successful measurement of the functional activity of cystatin C in the CSF, and show that total cystatin C activity can be inferred from its total protein concentration. Our results also suggest that cystatin C is the major cysteine protease inhibitor in human CSF and altered CSF cystatin C concentration may play a role in the pathobiology of ALS and other neurological diseases. Cystatin C is a low molecular weight cysteine protease inhibitor that modulates protein degradation and cell-matrix interactions . This prin vitro and in vivo models. Therefore, cystatin C may have multiple functions relevant to the pathogenesis and progression of neurological disease. However, the influence of altered cystatin C levels in this context remains unclear.We and others have reported that cystatin C levels are reduced in the CSF of patients with amyotrophic lateral sclerosis (ALS) when compared to both healthy controls and neurological disease controls -9. Alterin vivo, resulting in activity differences that are either magnified or diminished relative to concentration differences. Along different lines, it is also possible that the post-translational modification or proteolytic processing of cystatin C is altered in some disease states, potentially resulting in altered baseline activity kinetics for the protein.While the alteration of CSF cystatin C concentration in various neurological disorders has been established, the relationship between the concentration and functional activity of this protein within the CSF is unknown. It is possible that CSF cystatin C exhibits simple, linear activity kinetics at all physiologic concentrations, such that it’s mean activity is a constant multiple of its total concentration. Alternatively, cystatin C may demonstrate concentration-dependent activity kinetics Once the concentration-to-activity relationship is clarified, it will be possible to draw conclusions about the potential effects of altered cystatin C concentration in neurological disease states. If the observed differences in CSF cystatin C concentration do result in significant changes in its cysteine protease inhibitory activity, this could directly impact the protein milieu of the CSF, alter the extracellular matrix within the CNS, and influence downstream signaling events, potentially generating either neurotoxic or neuroprotective effects.To determine the relationship between cystatin C concentration and functional activity within the CSF, we modified a papain inhibition assay to enable the measurement of the protease inhibitory activity of cystatin C in this biofluid. We used this assay to evaluate cystatin C activity in CSF samples from a total of 69 ALS patients, healthy controls, and disease controls. We found that the functional activity of cystatin C within the CSF is directly related to its protein concentration, and that reduced cystatin C protein levels in the CSF of ALS patients likely correspond to reduced cysteine protease inhibitory activity. Our results provide an assay to measure cystatin C activity in CSF and suggest a functional consequence of reduced CSF cystatin C levels in ALS patients.For this study, we selected 69 independent CSF samples that were recently utilized in cystatin C biomarker discovery efforts [The total cystatin C concentration in each of the 69 CSF samples was previously determined by ELISA [For these experiments, the single CSF sample with the highest concentration of cystatin C was used. Immunoprecipitation (IP) of cystatin C from CSF was performed using a magnetic bead system . First, varying amounts  of affinity-purified goat anti-human cystatin C polyclonal antibody  were added to 18 μL aliquots of CSF and the solutions were briefly mixed at room temperature. Next, 100 μL of Protein A MicroBeads were added to each aliquot, and the solutions were mixed and incubated for 30 min on ice. During this incubation, the μ columns  were each primed with 200 μL of lysis buffer  and rinsed with 100 μL of assay buffer (see Papain inhibition assay below). Next, the CSF solutions were applied to the μ columns and the "flow-through" was collected. An additional 30 μL of assay buffer were then added to flush the remaining CSF solutions into the same flow-through collection tube. The columns were each rinsed with 50 μL of PBS and the combined flow-through solutions from all three steps were saved. The columns were then rinsed with an additional 750 μL of PBS and 100 μL of low-salt wash buffer . To elute the isolated cystatin C, 20 μL of pre-heated 1X SDS gel loading buffer were added to each μ column and incubated for 5 min at room temperature. Next, an additional 50 μL of pre-heated 1X SDS gel loading buffer were added and the elution products were collected. For subsequent IPs of flow-through solutions, 4 μL (0.8 μg) of anti-cystatin C antibody and 100 μL of MicroBeads were added to each flow-through sample, and the remaining steps were repeated as above.IP elution products were separated by electrophoresis on a NuPAGE® Novex® 12% Bis-Tris polyacrylamide gel , with 20 μl per gel lane. The proteins were transferred onto a PolyScreen polyvinylidene difluoride membrane  and blocked with 5% nonfat milk in 1X TBS/0.05% Tween-20 (TBST) for one hour at room temperature. A mouse monoclonal anti-human cystatin C primary antibody  was added to the blocking solution at a 1:1000 dilution and incubated overnight at 4°C. The membrane was thoroughly washed with TBST, and then a goat anti-mouse horseradish peroxidase-conjugated secondary antibody  was diluted by 1:5000 in 5% nonfat milk in TBST and applied to the membrane for two hours at room temperature. The membrane was thoroughly washed with TBST, and then the labelled proteins were visualized using Chemiluminescence Reagent Plus .The procedure we developed for this study was modified from an R&D Systems activity assay protocol for recombinant human cystatin C protein . Plant-derived papain  was diluted to 100 μg/mL in ice-cold activation buffer  and incubated at room temperature for 15 min. During this incubation, CSF samples from each patient were diluted at a 2:1 ratio by volume in either assay buffer  or polyclonal cystatin C blocking antibody  and incubated with gentle agitation for five min at room temperature. The CSF sample solutions were then further diluted 1:8 in assay buffer, and the activated papain solution was further diluted in assay buffer to 2 ug/mL. Equal volumes of the CSF and papain solutions were mixed and incubated at 37°C for 10 min. We next diluted the mixture 1:5 in assay buffer, and 50 μL were added to each well of a 96-well plate. 50 μL of ZPheArgAMC  substrate  was rapidly added to each well, and the plate was immediately inserted into a plate reader. The plate was read for 5 min in kinetic mode, using excitation and emission wavelengths of 380 nm and 460 nm, respectively.Samples were randomized to plates and plate positions, and assayed in triplicate. Each sample, and its corresponding sample/antibody solution, were always assayed in adjacent wells. A standard curve and an internal standard of mixed CSF were also included on each plate for quality control. The experiment was repeated with samples in reverse order on each plate to control for slight assay differences based on plate position. All experiments were performed at least twice.p < 0.05.All statistics were carried out using SPSS software. Data normality was assessed by the Kolmogorov-Smirnova and Shapiro-Wilk tests, and normality was only assumed when indicated by both tests. All calculated p-values were two-tailed, and the significance level was set at For each experiment, the reaction rate for each well was determined as slope of best-fit line of graphed RFU data. Intra-assay coefficients of variation (CVs) were calculated using the three reaction rates for each sample, and each sample/antibody mixture, on each plate. Data from one of the triplicate well readings was dropped from the analyses when it differed from both other wells by a CV ≥ 40, and the other two sample wells differed from each other by a CV less than half of the average CV between the outlier and the similar wells. This eliminated only 0.65% of the assay data points (6 of 930 data points). The average reaction rate for each raw CSF sample (without blocking antibody) was recorded as the total assay activity for that sample. The total assay activity was then subtracted from the average reaction rate for the same sample containing blocking antibody to determine the total cystatin C activity for that sample.All experiments were performed in triplicate and each experiment repeated at least twice. Inter-assay CVs were also determined for each sample using the total assay activity and, separately, the total cystatin C activity calculated for each separate experiment using the same guidelines described above.Coefficients of variation between duplicate/triplicate wells for each sample run, and between mean cystatin C activity plate results for each sample, were recalculated after dropping outlier data as described above. Because both groups of CV data failed normality, median values were calculated as the measure of central tendency. The overall intra-assay CV was calculated as the median of the individual CVs for all sample-runs. The CVs were non-normally distributed (range: 0.1–29.9%), and the median (3.6%) was less than the generally acceptable value for intra-assay variability (CV < 10%). Similarly, inter-assay CVs were determined using the final cystatin C activity level determined in each replicate experiment for each CSF sample. The CVs were non-normally distributed (range: 0.2–32.1%), and the median (9.1%) was well within the acceptable range for inter-assay variability (CV < 20%) .The correlations between cystatin C concentration and both assay activity and cystatin C activity in all samples as a combined group were assessed using non-parametric statistics because the data for total assay activity were non-normally distributed. For these analyses, SPSS software was used to calculate both the Spearman correlation coefficient and the two-tailed significance level. Identical methods were used to assess the same correlations in each diagnostic group because cystatin C concentrations in ALS patients also failed normality testing.Because cystatin C activity ratios were normally distributed in all three experimental groups and there were no significant differences among group variances, we compared group means by one-way ANOVA.For our final comparison of cystatin C concentration, total assay activity, and total cystatin C activity between groups, all diagnostic group data were normally distributed except for cystatin C concentration. For each data category, differences among diagnostic group means were identified by one-way ANOVA.Samples in all three diagnostic groups were paired by cystatin C concentration, and the seven samples in each group which could not be paired with samples from the other groups were dropped from this analysis. All paired data for cystatin C concentration, total assay activity, total cystatin C activity, and cystatin C activity ratio were normally distributed. Therefore, we were able to use the SPSS general linear model for repeated measures to assess differences in each of the four variables among diagnostic groups.To measure cystatin C protease inhibitory activity in the cerebrospinal fluid (CSF), we utilized a papain inhibition assay. Papain is a plant-derived cysteine protease that can hydrolyze the Arg-AMC amide bond of a Z-Phe-Arg-AMC substrate to release the fluorescent 7-amino-4-methyl coumarin (AMC) group. Relative fluorescence units (RFU) are then measured over time to determine the total papain activity. When a cysteine protease inhibitor (CPI) is added to the assay solution, it inhibits papain-mediated Z-F-R-AMC substrate cleavage, and fluorescence emission is reduced. The total activity of the CPI can then be estimated as the reduction in papain-mediated assay activity.There are several challenges when using this assay to measure the activity of a specific CPI within a biofluid sample, such as CSF. First, because CSF contains several CPIs, the total assay inhibition is a measure of the combined CPI activity and cannot be attributed to a single protein. Additionally, CSF also contains endogenous cysteine proteases that could potentially cleave the Z-F-R-AMC substrate and supplement papain activity. This would increase the overall fluorescence emission and decrease the apparent CPI activity determined by the assay.To control for these factors, we determined the total cystatin C activity in CSF by assaying both raw CSF, and CSF from the same patient sample that had been pre-incubated with an antibody that blocks cystatin C activity. For raw CSF samples, the total measured assay activity included the supplementary effects of all endogenous cysteine proteases as well as the inhibitory effects of all CPIs present in the sample. For the CSF treated with the blocking antibody, the measured assay activity included all of the factors present in raw CSF except for the inhibitory activity of cystatin C. Therefore, the total inhibitory activity of cystatin C in each sample could be calculated by subtracting the total assay activity for raw CSF from the total assay activity for CSF treated with the blocking antibody.Experiments were first conducted to test the effectiveness of the polyclonal anti-cystatin C antibody in both binding to human cystatin C within CSF and in eliminating cystatin C activity from CSF samples. First, varying amounts of the antibody were used to immunoprecipitate (IP) cystatin C from the CSF. Western blots were then performed to assess the relative amounts of cystatin C removed from each sample aliquot by IP, as well as the amount remaining in each sample Figure . We founNext, CSF aliquots from the same sample were pre-incubated with varying amounts of the same polyclonal "blocking" antibody and used in the papain inhibition assay as described in Methods. We observed an antibody dose-dependent elimination of cystatin C inhibitory activity, with full elimination of function achieved by a sample to antibody ratio of 2:1, by volume in a CSF sample that contained the highest amount of cystatin C protein as determined by ELISA (Table p < 0.001 and Spearman r values between -0.86 to -0.91 for each individual subject group cystatin C and total papain assay activity.This 2:1 sample to antibody ratio was used to perform the papain inhibition assay in all CSF samples to determine cystatin C functional activity in each sample. Before separating the data into diagnostic groups, we graphed both total assay activity and total cystatin C activity versus the cystatin C concentration for each sample. We observed a strong, indirect relationship between CSF cystatin C concentration and total papain assay activity (Figure We next compared CSF cystatin C concentration and activity levels among the three diagnostic groups Table . The meaFinally, to fully control for differences in mean cystatin C concentration among diagnostic groups, we paired select data from each group for statistical comparison. Subsets of samples with matched cystatin C concentrations were selected from each group, generating three diagnostic subgroups with identical mean cystatin C protein concentrations Table . We thenPrior studies have reported that cystatin C concentrations are decreased in the CSF of ALS patients relative to healthy controls, but it has been unclear if these reductions in protein level result in proportional reductions in cysteine protease inhibitory activity. To address this important question, we modified a papain inhibition assay to allow for the direct measurement cystatin C functional activity in human CSF. The assay performed with low inter- and intra-assay variability, and allowed the reliable evaluation of cystatin C activity in multiple CSF samples from ALS patients and controls. Notably, this assay measures only the activity of cystatin C at its primary active site, which interacts with papain-like cysteine proteases. Therefore, the experimental findings of this study only apply to the activity of cystatin C against its primary ligands, and do not address its interactions with legumain-like cysteine proteases.As expected, cystatin C concentration demonstrated a strong, direct relationship with the magnitude of its inhibitory activity Figure . This coCystatin C concentration also demonstrated a strong, indirect relationship with the overall activity of the papain assay Figure . This inWhen the data were divided into diagnostic groups, comparable correlations were maintained between cystatin C concentration and both its own activity and total papain assay activity in each group Table . Activitin vivo[in vitro[Reduced CPI activity within the CSF of ALS or other neurologic disease patients may contribute to either toxic or protective pathways related to disease pathogenesis. Cystatin C has been reported to exhibit neurotoxic effects when injected into the rodent brain , and whein vivo and in v[in vitro, and pot[in vitro. The pro[in vitro,24, secr[in vitro, and rel[in vitro-29. TherExtracellular cystatin C also may be internalized by motor neurons and/or glial cells, and subsequently affect intracellular processes. Efficient cystatin C uptake has been demonstrated with multiple non-neuronal human cell lines . If thisWe have determined that the functional activity of cystatin C within the CSF can be effectively measured by a papain inhibition assay, and that CSF cystatin C activity is directly proportional to its concentration in ALS patients and controls. Our data indicates that cystatin C is the predominant cysteine protease inhibitor in the human CSF.Robert Bowser is co-Founder of Knopp Biosciences LLC and Iron Horse Diagnostics Inc., biotechnology companies devoted to the development of diagnostics and therapies for ALS and other neurological diseases.Conceived and designed the experiments: MW and RB. Performed the experiments: IB and MW. Analyzed the data: MW. Wrote and revised the paper: MW and RB. Edited and revised the paper: IB. All authors read and approved the final manuscript.


Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein. Knowledge of genes that are specific to humans is likely to shed light on our understanding of human physiology and pathology. TBC1D3 is a hominoid-specific gene that maps to human chromosome 17 In this study, we focused on the role of TBC1D3 in regulating the mTOR-S6K-IRS-1 axis within the insulin signaling pathway. Because of its central role in cell proliferation and glucose homeostasis, the signaling pathway controlled by insulin and other growth factors is one of the most intensely investigated cellular pathways. Insulin resistance precedes the development of type 2 diabetes, and in addition, hyper-activation of the pathway is associated with several proliferative diseases Insulin receptor signaling is mediated by the recruitment of numerous adapters and among the most important is IRS-1. IRS-1-mediated insulin signaling is attenuated by IRS-1 ubiquitination and degradation In mammalian cells, at least two proteins are regulated by the activity of mTOR: 4E-BP1 and S6K, which are involved in the initiation of protein translation and are regulated by Ser/Thr phosphorylation Using insulin-sensitive human cell lines, we report that TBC1D3 expression suppresses IRS-1 ubiquitination and delays degradation by selectively enhancing the dephosphorylation of S6K at T389. We provide evidence that a specific PP2A regulatory subunit B56γ, recently shown to dephosphorylate S6K at T389 This study was aimed at exploring the effect of TBC1D3 on insulin/IGF-1 signal transduction pathway. Our findings indicate that TBC1D3 expression enhances the intensity and duration of the insulin/IGF-1 signaling cascade.In previous studies, we demonstrated that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) To exclude misleading phenotypes due to over-expression, we utilized RNA interference to suppress endogenous TBC1D3. Two different siRNAs directed against TBC1D3 (siRNA#1 and siRNA#2) were transfected into HepG2 cells. A scrambled, irrelevant siRNA (scr) was used as a negative control. 36 hours later, cells were serum-starved and stimulated with insulin (10 nM).We were not able to detect the expression of endogenous TBC1D3 protein, since a reliable anti-TBC1D3 antibody is currently unavailable. Thus, a quantitative real-time PCR was carried out to determine the efficiency of siRNA-mediated target suppression. Both siRNA duplexes decreased mRNA levels of TBC1D3 by 60%–70% (data not shown). No off-target silencing effect was observed.Consistent with the results obtained when over-expressing TBC1D3, silencing of TBC1D3 abrogated the activation of Akt following insulin stimulation . CollectThe insulin receptor (IR) is activated by autophosphorylation on tyrosine residues that function as docking sites for adaptor proteins. Among these are the insulin receptor substrate (IRS) proteins. IRS-1, which is widely expressed, is the most-well-characterized member of the IRS family. IRS proteins are rapidly recruited to the insulin receptor and activated by tyrosine phosphorylation following insulin stimulation To gauge the effect of TBC1D3 expression on IRS-1 phosphorylation, IRS-1 and myc-TBC1D3 were co-expressed in Hek293 cells . Total cell lysates were prepared after insulin treatment and IRS-1 phosphorylation status was analyzed by Western blotting using phospho-specific antibodies. Total IRS-1 levels were measured and used to normalize and quantify the results in each sample.Over-expression of TBC1D3 did not affect insulin-stimulated IRS-1 phosphorylation on tyrosine residues (data not shown), however it led to a reduced phosphorylation of recombinant IRS-1 on S307, S312 and S636/639 in response to insulin, when compared to control cells . These oA recent publication by Ye and colleagues, found IRS-1:S270 as a novel IRS-1 phosphorylation site that may be involved in regulating the phosphorylation of other serine residues by TNF-α de novo IRS-1 synthesis.To understand the functional consequences of decreased IRS-1 serine phosphorylation by TBC1D3 over-expression, we next studied the kinetics of IRS-1 degradation. To test whether TBC1D3 delays this process, DU145 cells  transiently transfected with myc-TBC1D3 or empty vector were serum-starved, and incubated with insulin for the indicated times. Total cellular levels were quantified by Western blotting and were used to normalize the amount of protein remaining at each time point. Cycloheximide (25 µg/ml) was added to block Control cells showed a rapid degradation of IRS-1; about 50% of the IRS-1 signal was lost after 2 hours of insulin treatment . HoweverIn agreement with these results, over-expression of TBC1D3 has also resulted in suppression of IRS-1 ubiquitination (data not shown), suggesting that TBC1D3 regulates IRS-1 turnover via modulation of its ubiquitination status.mTOR is a serine/threonine protein kinase that regulates cell growth and proliferation through a rapamycin-sensitive pathway involving the regulatory proteins p70 S6K and the eukaryotic initiation factor 4E-binding protein-1 (4EBP1) To support this hypothesis, we next determined whether mTOR, the kinase responsible for S6K activation, was defective by evaluating the phosphorylation status of the other mTOR substrate, 4EBP1. However, phosphorylation of 4EBP1 was normal and similar in both TBC1D3-expressing and control cells after insulin treatment . Moreove50 = 0.51 nM) Reduced S6K phosphorylation in cells expressing TBC1D3 could be due to decreased mTOR kinase activity or increased phosphatase activity. Several recent studies have linked PP2A to the regulation of S6K in vitro using a specific synthetic phosphopeptide  but TBC1D3 expression was less effective in reducing S6K:T389 phosphorylation.3E. We examined the effect of TBC1D3 on IRS-1:S636/639 phosphorylation in cells transfected with IRS-1 and with either TBC1D3 alone or TBC1D3 and S6K-ED3E. Following stimulation with insulin, IRS-1 was immunoprecipitated, resolved by SDS-PAGE and blotted with the specific antibody against phospho-IRS-1:S636/639. The results are presented in 3E construct, expression of TBC1D3 was unable to suppress insulin-stimulated IRS-1:S636/639 phosphorylation indicating that S6K is the likely target of TBC1D3.To confirm the role of S6K in TBC1D3-suppression of IRS-1 phosphorylation, we took advantage of a rapamycin-resistant mutant of S6K, S6K-EDWe speculated that TBC1D3 modulates the effect of PP2A B56γ on S6K:T389 phosphorylation by functionally interacting, directly or indirectly, with PP2A B56γ. To verify this possibility, a reciprocal co-immunoprecipitation experiment was carried out to determine whether TBC1D3 interacts with endogenous B56γ. Hek293 cells transfected with empty vector or myc-TBC1D3 were lysed and immunoprecipitated with anti-myc or anti-B56γ antibodies followed by SDS-PAGE and Western blotting. As shown in et alet alet alet alet alTBC1D3 appears to have evolved along the hominoid-lineage 35 million years ago by segmental duplication There has been a growing realization that signaling abnormalities within nutrient signaling pathways can lead to abnormal cell growth and cancer. The insulin/IGF-1 signaling pathway and the mTOR/S6K cascade defines a metabolic network that integrates glucose metabolism and energy balance with cell proliferation and survival We report that TBC1D3 expression enhances the signaling properties of insulin receptor as determined by standard assay, the phosphorylation of Akt. Importantly, silencing of TBC1D3 suppressed the activation of Akt by insulin. Degradation of endogenous IRS-1 was suppressed following insulin treatment in cells expressing TBC1D3 . Since sIRS-1 degradation following ubiquitination is part of the negative feedback loop designed to initiate and limit nutrient uptake and growth In their pioneering study, Pan and colleagues To explore the mechanisms of TBC1D3-induced suppression of IRS-1 phosphorylation, we focused on the effect of TBC1D3 on S6K activation. S6K is activated by mTOR signaling via phosphorylation on T389 and inactivated by PP2A-dependent dephosphorylation. Expression of TBC1D3 can suppress phosphorylation and/or enhance dephosphorylation of S6K at T389.To test the hypothesis that TBC1D3 enhances the dephosphorylation and inactivation of S6K in PP2A-dependent manner, we took advantage of the potent PP2A inhibitor OA. OA specifically inhibits the protein phosphatases 1 (PP1) and 2A (PP2A) et alin vivo. The assembly and function of the active PP2A complex is regulated by methylation PP2A is a large family of heterotrimeric enzymes that account for the majority of total serine/threonine phosphatase activity in most tissues and cells et alPPP2R5C, encoding the B56γ regulatory subunit of PP2A, acts as a specific negative regulator of S6K phophorylation in flies and humans. This discovery prompted us to examine to role of PP2A B56γ in TBC1D3 suppression of S6K activation. As shown in A provocative study by Hahn 3E 3E permitted the insulin sensitive stimulation of IRS-1:S636/639 phosphorylation but abolished the TBC1D3 inhibitory effect. Clearly, other unknown factors are at play such as the effect of TBC1D3 expression on overall PP2A activity , we took advantage of a rapamycin-resistant mutant of S6K called S6K-EDactivity . Human Pactivity .Taken together, the reversal of TBC1D3-enhanced S6K phosphorylation by OA and by silencing PP2A B56γ supports the conclusion that PP2A B56γ is a prime downstream target of TBC1D3. Based on these early observations, we offer a working model  to guideNotably, previous work showed that TBC1D3 also suppressed the recruitment of the E3 ligase CBL to the EGF receptor thereby suppressing the ubiquitination and degradation of the activated EGFR The human genome encodes a significant number of genes that are either human- or hominoid-specific and that have no known orthologs in lower organisms 3E was a gift from Dr. Tony Hunter, Salk Institute  Monoclonal α-tubulin, human insulin and MG132 were purchased from Sigma-Aldrich . Antibodies against PKB/Akt (Akt) as well as phospho-Akt S473, phospho-MAPK, total MAPK, phospho-4EBP1, phospho-S636/639, phospho-S1101 and phospho-S307 of IRS-1, S6K and phospho-S6K were obtained from Cell Signaling Technology . Phospho-S270 of IRS-1 and PI3k-p85a antibody were obtained from Santa Cruz Biotechnology . Antibody to phospho-S312 of IRS-1 was acquired from Biosource . Monoclonal antibody against phospho-Tyr of IRS-1 was purchased from BD Transduction Laboratories . IRS-1 antibody was provided by Dr. Mike Mueckler, Washington University . The monoclonal antibody directed against the C-terminal 50 amino acids of TBC1D3 was generated by the Hybridoma Center, Washington University . A rabbit polyclonal antibody against PP2A B56γ3 was a gift from Dr. Bill Hahn, Dana Farber Cancer Institute . pCLS2-IRS-1 construct was a gift from Dr. Jianping Ye, National Institutes of Health . Rapamycin was purchased from Enzo Life Sciences . pRK7-HA-S6K-ED5′- GCATCGACCGGGACGTAAG- 3′; reverse primer 5′- CCTCCGGGTTGTACTCCTCAT -3′, corresponding to nucleotide 431 and 550, respectively), using Platinum Taq DNA Polymerase Hi-Fi . Real-time PCR was carried out using an ABI Prism® 7000 Sequence Detection System . Cycling conditions were as follows: denaturation (95°C for 5 min), amplification and quantification , repeated 40 times. Each sample was run in triplicate.PP2A B56γ was successfully suppressed using three different shRNAs against PP2A B56γ  To obtain cell lysates, cell monolayers were washed with PBS and lysed in ice-cold lysis buffer . Cell lysates were clarified by centrifugation and proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes, which were blocked and probed with the indicated antibodies. To determine relative protein amounts, three representative exposures for each sample were quantified using AlphaEaseFc software .Protein phosphorylation and IRS-1 degradation were measured in cells serum-starved for 5 hours. Cells were incubated in the presence of insulin (10 nM) at 37°C, washed with ice-cold PBS and lysed. Proteins were separated by SDS-PAGE and analyzed by Western blotting.Cells were lysed in ice-cold immunoprecipitation buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM NaF, 1 mM sodium orthovanadate plus protease inhibitors. Extracts were immunoprecipitated by incubation with the appropriate antibody followed by immobilization on Protein G-Sepharose beads (Sigma). Beads were resuspended in sample buffer and analyzed by SDS-PAGE and Western blotting.PP2A activity was assayed using PP2A Immunoprecipitation Phosphatase Assay Kit (Upstate). Briefly, PP2A catalytic (C) subunit was immunoprecipitated from 300 mg of cell lysates using anti-PP2A C subunit antibody . Enzymatic activity was measured by incubation of the immunoprecipitated PP2A C subunit with the synthetic phosphopeptide K-R-pT-I-R-R following manufacturer's instructions.All experiments presented were repeated a minimum of three times. Results were expressed as mean values ± standard error (mean ± SE). Student's t-test or one-way analysis of variance (ANOVA) was performed to calculate statistical significance.


For the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible.The nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human FcεRI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1 : 100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen.P = 0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well .Sensitization with 15 pg/ml IgE was sufficient to detect IgE crosslinking–induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1 fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.The EXiLE test using RS-ATL8 cells is a promising  The CAP test is a highly automated, convenient and very sensitive method (sub ng/ml) for detecting serum IgE binding to allergens , were required to measure meaningful responses. These compounds have been reported to potentiate the degranulation of mast cells  following the manufacturer's protocol. Stable transfectants were selected using 600 μg/ml hygromycin. The RS-ATL8 cell line, the highest luciferase responder subclone after stimulation with 10 nM phorbol myristate acetate and 10 μM ionomycin, was established by limiting dilution and was grown in minimum essential medium (MEM) (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum , penicillin/streptomycin, GlutaMAX-I, 1.2 mg/ml geneticin, and 200 μg/ml hygromycin (Invitrogen).RBL-SX38 cells, expressing the human FcεRI α/β/γ-subunits, were a kind gift from Dr Kinet at Beth Israel Deaconess Medical Center , and were maintained as previously reported . The NFAPatients with suspected egg allergy referred to our hospital for investigation were enrolled in the study. Most of the patients had atopic dermatitis and asthma. Informed consent was obtained from patients, their parents, or both to collect and investigate serum samples further. The study was approved by the institutional review board of National Institute of Health Sciences and Ethics Committee of Fujita Health University School of Medicine. All sera were collected before food challenge to measure total and allergen-specific IgE levels using ImmunoCAP. Every patient was subjected to double-blind, placebo-controlled food challenges with heated egg and was judged positive if he or she was unable to eat the equivalent of a whole boiled egg, as described previously . Pooled 4 cells/50 μl/well) were plated onto a clear-bottom white 96-well plate , and were incubated for several hours at 37°C in a 5% CO2 incubator. Then, 5 μl of partly diluted (11 : 100) or serially diluted sera in MEM containing 10% FCS was added to the supernatant . After overnight incubation, the cells were washed once with sterile PBS and then stimulated for 3 h at 37°C in a 5% CO2 incubator with allergens diluted in MEM containing 10% FCS (50 μl/well). After stimulation, 50 μl of luciferase substrate solution containing cell lysis reagent  was added to the cells, and chemiluminescence was measured using an EnVision multilabel plate reader (Perkin Elmer). Luciferase expression levels are represented as the fold increase of light units compared with the background expression, after subtraction of a blank control (without cells).RS-ATL8 cells . OVA (1 μg/ml) or goat anti-human IgE antibody  diluted in prewarmed PIPES buffer was added to the cells (50 μl/well). After incubation for 30 min at 37°C, supernatants were collected, and residual cells were lysed with 0.2% Triton X-100. Activity of β-Hexosaminidase in the medium and within the cells was determined by a fluorometric assay using 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide as a substrate . The reaction was stopped with 0.25 M glycine buffer after 30 min incubation at 37°C. The plate was read on a Fluoroskan Ascent plate reader  using 380 nm excitation and 440 nm emission filters. Degranulation levels are represented as follows; R is the degranulation level; Rsup is released enzyme activity in the supernatant; Rppt is residual enzyme activity within the cell; blank is the buffer control.Stimulation-induced β-hexosaminidase release from RBL cells was measured as described previously with some modifications . RBL-SX3PLUS  following the manufacturer's protocol, and an EL340 .Cytotoxicity of human sera on RS-ATL8 cells was measured by a lactate dehydrogenase (LDH) release assay using the Cytotoxicity Detection Kitanova followed by Dunnett's test compared with negative controls without human serum. Fisher's exact test was performed to analyze correlations between the OFC and EXiLE or CAP tests. Spearman's rank correlation test was performed to compare the EXiLE and CAP test.The cytotoxic effect of human serum was analyzed using a 1-way In the present study, we transfected an NFAT-responsive luciferase reporter gene into human FcεRI α/β/γ-expressing RBL-SX38 cells. We obtained 16 clones after hygromycin resistance selection, and found that clone number 8 was the highest responder to phorbol ester and ionomycin (data not shown). This clone was designated as RS-ATL8; RBL-SX38 cell stably transfected with NFAT-Luciferase clone 8. The cells were sensitized with serially diluted healthy donor's serum overnight, and then stimulated with 1 μg/ml goat anti-human IgE antibody for 3 h. A dose-dependent increase in EXiLE in the RS-ATL8 cells was observed . The ori2O or 5′-N-ethylcarboxamide, to be added to the medium (A/ml), and stimulated with 1 μg/ml of OVA and anti-human IgE for 30 min in PIPES buffer or for 3 h in 10% FCS-containing MEM, respectively , and 12 of 19 were positive in the EXiLE test. Among 12 OFC positive patients’ sera, 11 and 12 were predicted to be positive by EXiLE and CAP tests, respectively, while among 7 OFC negative patients’ sera, 6 and 1 were predicted to be negative by EXiLE and CAP tests, respectively. As revealed by Fisher's exact test, the OFC and EXiLE tests correlated well (P = 0.001687), whereas the OFC and CAP tests did not (P = 0.3684).Nineteen sera from suspected egg-allergy patients who had been diagnosed by the OFC test  with heated egg and also by the egg white–specific serum IgE test (CAP test) were subjected to the EXiLE test for comparison. RS-ATL8 cells were sensitized with 1 : 100-diluted patients’ sera, and stimulated with 0, 1, 10, 100, and 1000 ng/ml of EWP, and EXiLE by EWP was calculated by assuming the background expression with no allergen was 1.0, and was judged to be positive if the maximum EXiLE value (Max EXiLE) was ≥2.0. R = 0.9319, P < 0.001), and between the CAP (specific IgE concentration) and Max EXiLE values .The EXiLE scores 1, 2, 3, and 4 correspond to the minimum concentrations of responsive EWP; 1000, 100, 10, and 1 ng/ml, respectively, while 0 means a nonresponder. We analyzed the correlation between the values and scores of the CAP and EXiLE tests by a Spearman's rank test. R = 0.8561, P < 0.001) than that between CAP and Max EXiLE. These results seemed to be consistent with a previous study by Dibbern Jr. et al. , whereas the results with the CAP test had no significant correlation to the OFC test (P = 0.3684). The low correlation between the CAP and OFC tests here could be partially explained by the high sensitivity of the CAP test. In CAP tests, a class of 2 (≥0.70 UA/ml) is usually adopted for a cut-off point following the manufacturer's instructions. However, Ando et al.  was set as a cut-off in P = 0.009546). Therefore, the CAP test is so sensitive that it seems to be suitable for the first screening of allergen-specific IgE in the sera.As shown in o et al.  recentlya et al.  also repA/ml specific IgE. Another possibility is that other substances, like chemokines and cytokines, can induce histamine release from human basophils  of egg white–specific IgE, but can eat eggs with no allergic symptoms. With this serum, EXiLE levels were gradually increased to 1.6 depending on EWP concentration, but did not exceed the 2.0 cut-off level  second screening of allergen-specific IgE in the serum following CAP tests, (ii) cross-reactivity tests between known allergens and potential allergens (safety assessment of gene-modified crops), (iii) standardization of allergen extracts for clinical usage, (iv) screening of novel allergens and/or epitopes, (v) detection of small amounts of allergens in foods, (vi) epidemiological study of allergens using a serum bank, and (vii) high-throughput screening for the seed compounds of anti-allergic drugs.This novel