Self-association appears to be an isoform specific phenomenon, as AKAP7α did not associate with itself or with AKAP7γ. However, AKAP7γ did associate with AKAP7δ, suggesting the long isoforms of the AKAP can form heterodimers. Surface plasmon resonance found that the AKAP7γ self-association occurs via two high affinity binding sites with KD values in the low nanomolar range. Mapping of the binding sites by peptide array reveals that AKAP7γ interacts with itself through multiple regions. Photon counting histogram analysis (PCH) of AKAP7γ-EGFP expressed in HEK-293 cells confirmed that AKAP7γ-EGFP self-associates in a cellular context. Lastly, computational modeling of PKA dynamics within AKAP7γ complexes suggests that oligomerization may augment phosphorylation of scaffolded PKA substrates. In conclusion, our study reveals that AKAP7γ forms both homo- and heterodimers with the long isoforms of the AKAP and that this phenomenon could be an important step in mediating effective substrate phosphorylation in cellular microdomains.A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7 Phosphorylation of target proteins is a key mechanism utilized by the cell to induce changes in physiology and function. Numerous kinase-substrate interactions have been defined, yet the mechanism through which specificity of these phosphorylation events is achieved is still unclear. Recent work has suggested that the subcellular localization of signaling enzymes to discrete locations in the cell confers spatiotemporal control over phosphorylation events . This is Saccharomyces cerevisiae pheromone response pathway, dimerizes upon pheromone treatment, to enhance MAP kinase signaling in the cell  (Biacore amine coupling kit). The amount of ligand bound was 150 RU (resonance units). Protein analytes (AKAP7γ or AKAP7α) were diluted over a range of concentrations (12.5–200 nM) in HBS buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, and 0.005% Surfactant P20) and were injected over the sensor surface at a flow rate of 30 μL/min for 300 s. After injection phase, dissociation was monitored in HBS buffer for 300 s at the same flow rate. The surface was regenerated between injections using 10 mM NaCl at a flow rate of 50 μL/min for 30 s. Obtained sensorgrams were analyzed by Biacore T100 evaluation software.SPR analysis was performed using the Biacore T100 platform. Purified recombinant AKAP7γ was synthesized as 20-mer peptides with three amino acid offsets on membranes using a MultiPep automated peptide synthesizer . The peptide array membranes were blocked for 2 hours in 1% casein in TBST  before incubation with 2 µg/mL recombinant human S-tagged AKAP7γ in 1% casein (in TBST) overnight at 4°C. Thereafter the membranes were washed five times in TBST for 5 min before incubation with HRP-conjugated anti-S-tag antibody  for 1 hour at room temperature. Binding was detected using ECL Prime  and chemiluminescence signals were detected by Las 1000 .Human AKAP7β-mercaptoethanol at 60°C and washed three times in TBST for 10 min before being blocked in 1% casein (in TBST) overnight at 4°C. Concomitantly, 2 μg/mL of recombinant human His-S-AKAP18γ protein was preincubated with or without 10 µM phospholamban peptide (1–30) overnight at 4°C, before being overlaid onto the peptide array membranes for 2 hours at room temperature. The membranes were washed, incubated with HRP-conjugated anti-S-tag antibody, and developed as described above. The PLB (amino acids 1–30) peptide was synthesized with an N-terminal biotin tag and purified to 89.0% purity by GenScript Corp., Piscataway, New Jersey, USA. Biotin-phospholamban (PLB) peptide amino acids 1–30: MEKVQYLTRSAIRRASTIEMPQQARQNLQN.For the peptide competition assay, the membranes were stripped for 45 min in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM μs. Global analysis of measurements with count rates ranging from 20 to 100 kHz was performed for each construct. Monomeric and dimeric (tandem) EGFP histograms were fit using a single-component model, while AKAP7γ-EGFP histograms were fit using a two-component model with the brightness values constrained to those derived from the fit of the monomeric and dimeric EGFP. Measurements exhibiting photo-bleaching or other anomalies producing instability of the fluctuation trace were excluded from analysis. A single-photon three-dimensional Gaussian model was assumed for the excitation volume.Photon counting in live HEK-293 cells was performed with a Zeiss LSM 510 ConFocor 3 confocal microscope mounted on an Axiovert 200 M. Excitation was achieved at 488 nm with a 30 mW Argon laser at 50% power with attenuation set to 0.5. Fluorescence emission was collected with a 40x 1.2 NA water immersion objective (Zeiss C-Apochromat 1.2 W Corr UV-Vis-IR). Detection was performed with an avalanche photodiode (Zeiss). For each cell, five 10 s measurements were made from five different locations within a given cell. Photon counting histograms (PCH) were generated using LSM-FCS software with a bin time of 10 http://www.vcell.org). The model is composed of 18 species, three compartments , and 11 reactions. The reaction network can be considered as being composed of four general modules: (1) production and degradation of cAMP, (2) the dynamic interactions of regulatory subunit, catalytic subunit and cAMP in the cytosol, (3) the dynamics of these same proteins and cAMP scaffolded on the ER membrane by AKAP, and (4) phosphorylation/dephosphorylation of target protein X on the ER membrane. A diagram of the reaction network and table of the kinetic parameters may be found in the supplemental information. For each oligomeric state of the scaffold represented in the different versions of the model, the concentration of each species is initialized with a value near steady state in unstimulated conditions. Each simulation is allowed to run for 200 s before stimulation, thus allowing each species to reach its equilibrium constant for each oligomer. Stimulation of PKA activity is achieved by increasing the amount of AC present on the plasma membrane from 5/µm2 to 50/µm2 for a period of 100 s, simulating the increase in Gs-associated AC during β-adrenergic stimulation. The resulting increase in cAMP leads to activation of scaffolded PKA and phosphorylation of the target protein X. Active PKA is then inactivated by interacting with the PKA inhibitor PKI. The ability of RIIα to self-regulate via autophosphorylation has been shown previously  in the Henri-Michaelis Menten irreversible reaction step for phosphorylation of X:A compartmental deterministic model of scaffold oligomerization was implemented using the Virtual Cell modeling software  or MBP alone. The protein stain of the nitrocellulose before western blot analysis showing AKAP7γ-MBP (lanes 1 and 2) or MBP alone (lanes 3 and 4) is shown in μL) from cells expressing AKAP7γ-EGFP is shown in lane 5. Importantly, AKAP7γ-MBP was able to pull down AKAP7γ-EGFP from the transfected cell lysate  for each immunoprecipitation is shown in γ-mCherry and AKAP7γ-EGFP associate in cells (γ-mCherry (lanes 1 and 2). As a control, both EGFP (lanes 1 and 2) and AKAP7γ-EGFP (lanes 3 and 4) immunoprecipitates were probed with anti-GFP to demonstrate the immunoprecipitation of both proteins  or MBP alone (lane 3) incubated with bacterially expressed S-tagged AKAP7γ. The protein stain of the nitrocellulose membrane before western blot analysis displaying the MPB tagged proteins used is shown in γ input for each experiment is shown in the middle panel. Importantly, we detect a direct association between S-tagged AKAP7γ and AKAP7γ-MBP , but not S-tagged AKAP7γ and MBP . Based on these experiments, we conclude that AKAP7γ can stably associate with itself.In order to understand the molecular architecture of the AKAP7 AKAP7 gene encodes at least 4 alternatively spliced isoforms: α, β, γ, and δ [α  and  β) are lipid modified in their N-terminal domain, directing their location to the plasma membrane [γ  and  δ) require additional protein-protein interactions for their intracellular targeting [γ-EGFP and incubated with purified recombinant S-tagged AKAP7α or AKAP7γ bound to S-protein beads. The protein stain of the nitrocellulose membrane before western blot analysis, demonstrating that the beads are charged with AKAP7α (lanes 2 and 3) or AKAPγ (lanes 4 and 5), is shown in μL) from cells expressing AKAP7γ-EGFP is shown in lane 1. Importantly, S-tagged AKAP7γ was able to pull down AKAP7γ-EGFP specifically from the transfected cell lysate (γ-EGFP with AKAP7α pulldowns (α-EGFP was incubated with S-beads charged with S-tagged AKAP7α (lanes 2 and 3) or AKAP7γ (lanes 4 and 5). As expected, we did not detect any interaction between the two isoforms . AKAP7γ or AKAP7α was chemically conjugated in separate flow chambers on a CM5 sensor chip. A range of concentrations of either AKAP7γ (α (KD) was determined from the fit of the sensorgrams. We detected a high affinity interaction between AKAP7γ and itself that is best fit with a heterogeneous analyte model, suggesting at least two sites of interaction that display binding affinities of 0.537 nM and 79.7 nM (χ2 = 1.33)  or AKAPγ (lane 2) is shown in μL) from cells expressing AKAP7δ-YFP is shown in the middle panel. Importantly, AKAP7γ was able to associate with AKAP7δ. To determine if these two isoforms could associate in cells, we coexpressed AKAP7δ-YFP in the presence (lane 2) and absence (lane 1) of AKAP7γ-mCherry HEK-293 cells. AKAP7δ-YFP input (20 μL) for each immunoprecipitation is shown in γ-mCherry and AKAP7δ-YFP associate in cells  [γ can form both homodimers and heterodimers with the longer isoforms of the AKAP7 gene family.Our data so far suggests that the long isoform of the AKAP can form homodimers. Next we investigated if AKAP7/371626) . Taken tγ and itself. To determine the number and location of binding sites, a series of AKAP7γ fragments were created. The common PKA binding domain is maintained, contained in amino acids 29–42 of AKAP18α (γ-EGFP (μL) from cells expressing AKAP7γ-EGFP is shown in the designated lane. In confirmation of our previous results, recombinant purified full length AKAP7γ, but not AKAP7α, was able to pull down AKAP7γ-EGFP from transfected cell lysate  and AKAP7γ(150–323), were able to pull down AKAP7γ-EGFP from transfected cell lysate, revealing that each half of the protein participates in the homoassociation of AKAP7γ . Furthermore, a peptide consisting of amino acids 150–300 of AKAP7γ also did not significantly reduce the presence of full length AKAP bound to the AKAP7γ-MBP (lanes 6-7). However, using both peptides simultaneously did attenuate oligomerization (lanes 8-9). Quantification of the percent binding is shown in We next looked to see if any of these regions could be used to disrupt the oligomerization. We conducted a pulldown assay using amylose beads charged with bacterially expressed AKAP7γ binding partners, including PKC, PP1, the PP1 inhibitor I-1, phospholamban, PDE4D3, and PDE3A [γ. To begin, HEK293 cells were transfected with either full length AKAP7γ-EGFP or the one lacking the PKA binding domain (AKAP7γ-ΔPKA-EGFP). Isolated lysate was incubated with purified recombinant S-tagged full length AKAP7γ or AKAP7γ lacking the PKA domain (AKAP7γ-1-268) bound to S-protein beads. The protein stain of the nitrocellulose before western blot analysis demonstrating that the beads are charged is shown in μL) from cells is shown in the middle panel. As shown in γ was able to pull down AKAP7γ-ΔPKA-EGFP from cells  [γ-mCherry was incubated with purified recombinant S-tagged AKAP7γ bound to S-protein beads. The protein stain of the nitrocellulose before western blot analysis demonstrating that the beads are charged AKAPγ is shown in μL) from cells expressing AKAP7γ-mCherry is shown in the middle panel. Importantly, incubation of either AKAPIS or a control, scrambled peptide, did not disrupt AKAP7γ homoassociation.Previous work has defined many AKAP7nd PDE3A , 20–24. om cells , lane 1,(AKAPIS) . Lysate γ or His-S-AKAP7γ and PLB (1–30) onto 20-mer AKAP7γ fragments as in γ does not interfere with homodimerization. Taken together, the data presented in γ.Next we looked to see if the phospholamban binding domain, contained in amino acids 124–220, was involved in directing the interaction. Peptide array studies were conducted by overlaying either His-S-AKAP7γ with itself suggests the potential to form multimeric structures. This possibility was investigated via photon counting histogram (PCH) analysis of AKAP7γ-EGFP expressed in live HEK-293 cells. Photon counting histogram analysis is a method of fluorescence correlation spectroscopy that allows for determination of the size  and concentration  of given oligomer from fluctuations in signal intensity. This technique uses standard confocal optics to create a measurement volume on the order of 0.5 fL. As fluorescent particles diffuse through the measurement volume, their signal is detected by a highly sensitive detector, such as an avalanche photodiode. The change in signal intensity is dependent upon the average number of particles within the volume (n) and the brightness of a given particle (ε). At low average particle concentrations, the change in fluorescence intensity resulting from a single particle entering or leaving the volume is readily detected, while at high concentrations this change is less detectable. Detection of the change in intensity over time yields a fluctuation trace. The fluctuation trace is divided into small increments of time (bins) and the signal intensity (counts) is averaged within each increment. The average signal for each bin is then plotted as frequency versus counts/bin to yield the photon counting histogram  or a two-component model (AKAP7γ-EGFP) yielding a brightness value (ε) and concentration for either one or two species, respectively. This type of fitting allows for the derivation of a single brightness value based upon the aggregate data from all measurements of a given fluorescent construct, rather than averaging individual values derived from each individual measurement. Global fitting of the EGFP monomer and dimer yielded brightness values of 9468 ± 72 (χ2reduced = 1.10) and 16967 ± 100 (χ2reduced = 1.14), respectively . Of 75 total measurements included in the analysis, dimer was detected in 52 measurements and in 42 measurements >10% of the total particles detected represent dimer  and catalytic subunits (Cα) in response to cAMP, both in the cytosol and on the endoplasmic reticulum (ER) membrane. We chose to model the dynamics of AKAP7γ at the ER due to recent work demonstrating localization of the scaffold at the ER in cardiac myocytes [α, as this is documented as a key component for activation of Cα when complexed with RIIα [α subunits within an oligomer increases as the total number of activated Cα subunits increases ((α subunits were modeled to participate in the phosphorylation of a generic target protein X (X → pX) existing on the ER membrane ((n = 1) is simulated, phosphorylation of our target protein occurs as it would if the feed-forward assumption is not made. Removal of the stimulus leads to dephosphorylation of pX and return to baseline. Oligomerization of AKAP7γ affects the baseline, magnitude, and speed of phosphorylation of protein X  while the magnitude change in pX increases by 76% in comparison to monomer only  does not indicate oligomerization of the AKAP [γ, which is predicted to exist in an unordered arrangement, according to FOLDindex©. Several AKAPs have a natively disorganized, unfolded configuration that is hypothesized to aid in the docking of a multitude of binding partners [γ may contribute significantly to its oligomerization. In fact, our peptide array analysis demonstrates that the majority of the interacting sequence from 43 to 98 is not contained in the original crystal structure. Other possible mechanisms include posttranslational modification driven interactions and coiled-coil interactions [γ sequence using the PredictProtein website as well as both MARCOIL and LOGICOIL web resources supports these possibilities for AKAP7γ. Testing these potential mechanisms for oligomerization will help in delineating the means of AKAP7γ complex formation.Several recent reports have demonstrated homooligomerization of AKAP5, AKAP12, and AKAP-Lbc , 12, 14.the AKAP . This stpartners . Howeverractions –38. Anal in vitro artifact, as no increase in stoichiometry was detected in vivo [γ does not display any known intrinsic enzymatic activity, suggesting that this mechanism is not applicable.Signaling proteins utilize oligomerization to enhance their known cellular functions. However, the consequences of AKAP oligomerization are still unclear but may impact multiple processes. Mass spectrometry analysis of the AKAP5 complex suggests that, upon dimerization, AKAP5 associates with four calcineurin phosphatase heterodimers and two calmodulin molecules . By incr in vivo . Oligome in vivo . Disrupt in vivo . Howeverγ oligomerization, we created a computational model of PKA scaffolding. Significant work has demonstrated that release of active Cα subunit from the PKA holoenzyme requires not only cAMP binding to the RIIα subunit but also autophosphorylation of RIIα [γ oligomer, release of a single Cα subunit can potentiate the release of additional Cα via phosphorylation of RIIα present in the oligomer. In the monomeric state, a single Cα can stimulate release of only one extra Cα subunit, but, in a dimeric or oligomeric state, release of additional Cα subunits is possible. This feedback loop ultimately increases substrate phosphorylation due to the increased probability of phosphorylation resulting from increased Cα release , it is noteworthy that the authors did not demonstrate that the truncated protein products do not associate with PKA. If a truncated version of AKAP7δ/γ is still expressed in the mouse, it would still bind all the other components of the complex and perhaps the balance of phosphorylation to dephosphorylation is changed. In an investigation that compared the effects of a complete knockout of AKAP5 versus the one that just deleted the PKA binding domain, significant decreases in substrate phosphorylation were only seen in the complete knockout [δ/γ that should still associate with the truncated protein may have a dramatic effect on phospholamban phosphorylation. Further investigation of this mouse model is warranted before concluding that AKAP7δ/γ anchoring to PKA is not important.The importance of AKAP7 to PLB phosphorylation and cardiac calcium dynamics has recently been called into question. Jones and colleagues created a mouse expressing a truncated AKAP7, consisting of deletion of exon 7 of theAP7 gene . This exknockout . This waγ signaling complex and predicted via computational modeling that dimerization of AKAP may impact phosphorylation of PKA substrates. As several PKA targets of AKAP7γ have been identified, including PDE4D, PDE4D3, the PP1 inhibitor I-1, and the cardiac calcium regulatory protein phospholamban, AKAP7γ oligomerization has the possibility of affecting phosphorylation of these endogenous substrates.We have discovered an additional component of the AKAP7Reaction Kinetic parameters for PKA dynamics used in the model are shown in the Supplemental Table 1.The network diagram designed to test our model is shown in Supplemental Figure 1.γ does not interact with any other AKAPs expressed in the heart. HEK293 cells were transfected with either mAKAP-EGFP or AKAP79-EGFP. Isolated cell lysates were subjected to pulldown assays with either AKAP7α-S-Tag or AKAP7γ-S-Tag before subjected to Western blot analysis to determine association.Supplemental Figure 2: AKAP7


To illustrate the hypoxia signal mediated by SnRK1.1, phosphoproteomics was used to identify not only the new SnRK1.1 putative targets related to submergence, but also to gain an insight into early hypoxia signalling events. SnRK1.1K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1K48M, mpk6, and PTP1S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence.SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant ( Owing to the limited availability of oxygen in water, plants cannot obtain sufficient energy from the oxidative phosphorylation of mitochondria to maintain their necessary functions, such as growth and defence responses to environmental changes, when submerged. Consequently, in order to avert an energy crisis during submergence plants have to adjust their metabolism. To maintain ATP production, plants switch to anaerobic fermentation. In addition, under hypoxia, plants also accumulate alanine and GABA through the GABA shunt. Alanine and GABA are used to store carbon and nitrogen which are easily lost during oxygen deprivation  of submergence in the dark. The transgenic lines, SnRK1.1T175A, SnRK1T175D, and SnRK1K48M in Col-0 were generated by T-DNA transformation containing the 35S promoter driving the ATP binding site mutant [SnRK1K48M] and catalytic site mutants [SnRK1T175D and SnRK1T175A] in the pMDC32 vector. We analysed SnRK1 expression and the submergence phenotype in seedlings of the T2 or T3 generations from at least two independent transgenic lines. The cMYC-SnRK1K48M overexpression lines were generated by transforming the 35S::cMYC-SnRK1K48M/pEarleyGate 203 vector into Col-0. The T-DNA insertion lines of mpk6 (SALK-127507) and ptp1 (SALK-118658C) were obtained from the ABRC, Ohio State University. The PTP1S7AS8A overexpression line was generated by transforming the 35S::PTP1S7AS8A-YFP/pEarleyGate 103 construct into ptp1.Transgenic lines expressing SnRK1−2 s−1)/8h dark cycle and placed vertically.All seeds were sterilized with 0.5% sodium hypochlorite for 10min and washed with sterilized water and then incubated with sterilized water at 4 °C in the dark for 3 d to achieve uniform germination. Seeds were sown on plates with 0.55% phytagel (Sigma-Aldrich) in half-strength Murashige and Skoog (MS) medium (Duchefa Biochemie) containing 0.5% sucrose at pH 5.7. The plates were transferred to a growth chamber at 22 °C with a 16h light (81 μmol m−2 s−1 at 22 °C) and 15h dark (at 18 °C). After the submergence treatment, plants were put back into a growth chamber under a 9h light (81 μmol m−2 s−1 at 22 °C) and 15h dark (at 18 °C) cycle for 4 d of recovery and then counted for damage. The percentage necrotic area of a leaf was used as the damage index. For the submergence treatment of 9-d-old seedlings, plates with plants on the surface of the medium containing half-strength MS salts and 0.5% sucrose at pH 5.7 were placed into half-strength MS liquid medium that was bubbled with 3% oxygen balanced with nitrogen for 0.5h, 1h, and 3h at room temperature in the dark. The MS liquid medium was pre-bubbled with 3% oxygen for 1h before use .For phenotypic assays, data were collected from 36 plants per genotype in six independent experiments. Four-week-old potted plants were put into distilled water at a depth of at least 5cm from the water surface in the dark in a growth chamber under a cycle of 9h light  from 9-d-old Col-0JXB onlineSupplementary Fig. S3 at . In brief, total protein was extracted from 9-d-old whole seedlings of Col-0 and SnRK1.1K48M treated with 0h, 0.5h, 1h, and 3h of submergence. The protein samples were reduced and alkylated by using dithiothreitol (DTT) and iodoacetamide (IAA). Subsequently, endoproteinase Lys-C (Wako) and trypsin (Promega) were added to digest the proteins into peptides. Then, the resulting peptide solution was acidified and desalted.Sample preparations of phosphoproteomics and proteomics were performed according to SnRK1.1K48M-5 that also underwent different durations of submergence were labelled 117, 118, 119, or 121, respectively. To reduce the peptide complexity of the eight samples, after labelling each reaction mixture was aliquoted into two parts of equal volume and these aliquots were mixed into three fractions as follows: fraction 1: 113,114,115, and 116; fraction 2: 117,118,119, and 121; fraction 3: 113,114,117, and 118. The profile of each fraction in different biological repeats is listed in JXB onlineSupplementary Table S1 at .For phosphoproteomics, samples of Col-0 that underwent different durations of submergence  were labelled 113, 114, 115, or 116, respectively, and samples from SnRK1.1K48M-5 with different treatments were labelled 117–121, respectively.For total proteome quantitative analysis, the desalted peptide samples from Col-0 with different treatments were labelled 113–116, and samples from 2 ratio in each sample. The median ratio of each sample was derived from the frequency distribution histograms of log2 protein/phosphorylated peptide ratios of each sample.Protein identification, phosphorylation modification, and quantitative analyses were analysed by the Proteome Discoverer software . MS data were searched against the Arabidopsis protein database, TAIR10, downloaded from the Arabidopsis Information Resource Center. The data were normalized with the value of the 113-labelled sample and the median of the logP <0.05) are marked as “nosig”. The time point data that were not detected or detected once in three batches are marked as NA (not available).In the phosphoproteome quantitative analysis, only unique peptides with a pRS score >50 were used for quantiﬁcation. To integrate fraction 1 and fraction 3 in each batch, the values of fraction 3 were normalized by the average value of 117/113 in fraction 2 to get the ratio of 118/113, 119/113, and 121/113. The peptides detected in two of the three biological experiments were selected for bootstrap statistical analysis  in 20 μl of kinase buffer for 1h at 30 °C. The kinase reactions were performed with or without λ phosphatase (New England Biolabs) for 1h at 30 °C. All reaction products were resolved in a Phos Tag SDS PAGE (Wako) and S-tag immunoblotting. The details of recombinant protein expression and in vitro kinase assay are provided in the Supplementary data.The recombinant proteins of F2KP, PENTA, eIFiso4G1, and PTP1 were purified from , SnRK1.1K48M-5, and SnRK1.1K48M-9 treated for different durations of submergence were re-suspended with 1ml 70% methanol with 12 μg ml–1 ribitol, and then sonicated for 30min at 4 °C. Subsequently, samples were centrifuged at the highest speed at 4 °C for 15min. The supernatant of each sample was collected and vacuum dried. The dried samples were then derivatized by bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) and analysed using a Pegasus 4D GCxGC-TOFMS system . The detailed conditions of GCxGC-TOFMS system are provided in the Supplementary data.The ground tissue (100mg) from 9-d-old whole seedlings of Col-0For the BIFC assay, the SnRK1.1, PTP1, and MPK6 coding regions were constructed into the destination expression vectors 3130 (pSAT-35S:: cYFP-DEST) and 3136 (pSAT-35S::nYFP-DEST), respectively. The resulting 3130-target and 3136-SnRK1.1 constructs were mixed and transfected into Arabidopsis leaf protoplasts . The transcripts of SnRK1.1 and SnRK1.2 were not changed under submergence  were generated. We selected two SnRK1.1K48M, two SnRK1.1T175A mutants, and one SnRK1.1T175D mutant that have different transcript and protein levels of the SnRK1 transgene, for further analysis . We first examined the submergence tolerance of these transgenic lines by subjecting 4-week-old plants to submergence for 2.5 d (60h) and recovery for 4 d. The percentage necrotic area of a leaf was used as the damage index (K48M and SnRK1.1T175A) were more sensitive to submergence (T175D) in Col-0 according to the results of .Prior studies indicated a potential functional redundancy of SnRK1.1 and SnRK1.2 in hypoxia responses  ≥50 that were identified in two out of three repeats were selected. A total of 615 unique phosphopeptides that belong to 485 proteins was identified by phosphoproteomics . Three steps were used to generate a list of up- or down-regulated phosphopeptides from these 615 peptides. (i) We normalized all the data with the value of Col-0 in normal conditions. (ii) Because the standard deviations of three biological repeats were E0.3 (Supplementary Table S1), the proteins with an average fold-change in phosphorylation level >1.3 or <0.7 and that passed the bootstrap analysis (P <0.05) were selected as being significantly different under submergence. This generated 57 ‘proteins with up-regulated phosphorylation’ (PUP) and 27 ‘proteins with down-regulated phosphorylation’ (PDP) in Col-0 under submergence . These proteins were mainly located in the nucleus and plasma membrane . We also identified 47 PUP and 60 PDP in SnRK1.1K48M-5 under submergence (Supplementary Table S1). (iii) Since we analysed the phosphorylated proteins profiles of Col-0 and SnRK1.1K48M-5 by LC MS/MS separately, it meant that the phosphorylation of some proteins was only detected in one of the lines. Therefore, these proteins were excluded from the lists of PDP and PUP. In addition, some phosphorylated proteins in SnRK1.1K48M-5 had pRS scores lower than 50, but not in Col-0, suggesting that their phosphorylation was disrupted by SnRK1.1K48M-5. We added these phosphorylated proteins to the PUP lists. Taken together, there were 50 PUP and 18 PDP in Col-0 and 29 PUP and 42 PDP in SnRK1.1K48M-5 under submergence  showed that 38 only appeared in Col-0, 12 appeared in both Col-0 and SnRK1.1K48M-5, and 17 appeared only in SnRK1.1K48M-5. These results suggested that 55 PUP were under SnRK1 regulation. Noticeably, the protein levels of 38 ‘Col-0 only PUP’ were not changed significantly in Col-0 and SnRK1.1K48M-5 under submergence , suggesting that SnRK1.1 only regulated their phosphorylation. On the other hand, in the analysis of PDP (Supplementary Table S1), there were 10 PDP in Col-0 only, eight in both Col-0 and SnRK1.1K48M-5, and 34 in SnRK1.1K48M-5 only. These results suggested that the phosphorylation of 44 PDP was under SnRK1 regulation. The protein levels of 10 PDP in Col-0 only were not changed significantly in Col-0 and SnRK1.1K48M-5 under submergence , revealing that SnRK1.1 was only involved in their phosphorylation reduction. Therefore, under submergence, 99 phosphorylated proteins, including 55 PUP and 44 PDP, were under SnRK1.1 regulation . To test whether they are direct targets of SnRK1.1, we selected four candidates in different functional categories, eIFiso4G1 (a translation initiation factor), PENTA (an RNA stability regulator), PTP1  , and F2KP, which is a well-known SnRK1.1 substrate, for the in vitro kinase assay. These proteins were fused with His and S tags and were obtained by E. coli expression systems and affinity-purification. In addition, the active SnRK1.1 and inactive SnRK1.1K48M were immunoprecipitated from Col-0 and cMYC-SnRK1K48M-1 that had been submerged for 1h. The specificity of the IP products was examined by SnRK1.1 immunoblotting following Phos Tag SDS PAGE . After mixing the four recombinant proteins with SnRK1.1 (IP-SnRK1.1-S) respectively, through S tag immunoblotting following Phos Tag SDS PAGE, the four recombinant proteins . F2KP is involved in glycogenesis (in vitro (Supplementary Table S5). This phosphorylation site was conserved in TPS5, 6, and 7 and was phosphorylated in response to glycolysis inhibitor (2-deoxyglucose) that stimulates the activity of SnRK1 (Supplementary Table S1), the other Class II TPS, TPS8, also had increased phosphorylation and was also phosphorylated at the consensus sequence of SnRK1 (phiXXXXS/TXXXphi), which is a 10-residue motif with phi being a hydrophobic residue , and X representing random amino acids . Alanine is accumulated, but α-KG is also used in the GABA shunt and non-circle TCA  of submergence , including PROTEIN TYROSINE PHOSPHATASE 1 (PTP1). PTP1 is a primary target of H2O2 (SnRK1.1K48M-5 is significantly lower than in Col-0 (Supplementary Table S5), and PTP1 was directly phosphorylated by SnRK1.1 in vitro . Through MS-MS analysis, the phosphorylation sites of PTP1 by the in vitro kinase assay were identified at the 6th threonine (PTPT6) and 7th and 8th serine (PTPS7 and PTPS8) (Supplementary Table S1), which were also identified in our phosphoproteomics study (Supplementary Table S1). We compared the Arabidopsis PTP1 sequence with orthologues from other plants (Glycine max) were serine or threonine, which can be phosphorylated. Interestingly, the phosphorylation possibility of PTPS8 is the highest in the in vitro kinase assay and two of three proteomic biological repeats. We therefore focused on the examination of phosphorylation of PTP1S7 and PTPS8.The other prominent group of putative SnRK1.1 substrates comprised proteins related to the stress response . We compared the MPK3/6 phosphorylation in Col-0, ptp1, and the two PTP1S7AS8A lines. MPK6 was phosphorylated in Col-0 after 30min of submergence, but the phosphorylation of MPK3 was not significant . However, the increases in MPK6 phosphorylation in the two PTP1S7AS8A transgenic lines were lower than in Col-0.  fused with mCherry was used as a nuclear marker and a transient effect control . Taken together, these results suggested that SnRK1.1 directly interacted with PTP1 to phosphorylate PTP1.We performed bimolecular fluorescence complementation (BiFC) assays to examine the interaction of SnRK1.1, PTP1, and MPK6. The C-terminal region of  control , lane 3 SnRK1.1K48M-5 . To validate that MPK6 signalling is affected in SnRK1K48M mutants, we compared the expression of predicted downstream targets of MPK6, HsfA2 and ZAT10  motif of MPK6 phosphorylation, revealing that the phosphorylation of MPK6T221 and MPK6Y223 was disrupted by PTP1 and SnRK1.1 under submergence. The TXY (X-D/E/P) motif is a dual phosphorylation site and phosphorylation of both residues is essential for the activation of the MPKs . It is known that eIF4A is phosphorylated within 20min of anoxia in maize and that eIF4A is an ATP dependent RNA helicase and regulates the eIF4G–eIF3 complex turnover in yeast  mRNA to regulate mRNA stability . PIP2 has been proposed to be an osmosensor , indicating that SnRK1 might be involved in regulating stomatal closure under submergence. Stomatal closure has been proposed to be a part of the hypoxia-triggered immunity response (2 influx into the shoots under submergence. Under submergence, there is a balance between O2 influx and consumption in the shoots. Plants can maintain the O2 level in the shoots by taking up O2 from stomata during dark periods or photosynthesis; it is then diffused to the roots through gas space diffusion (2 into the shoots in the early stage of submergence.In addition, AKS1 and AKS2, which are involved in ABA-dependent stomata closure (In summary, through our phosphoproteomic analysis, we discovered many SnRK1.1 downstream targets that participate in various biological processes . Among tJXB online.Supplementary data are available at Figure S1. The heterocomplex profile of SnRK1 under submergence.Figure S2. The profile of SnRK1 transgene lines.Figure S3. Flowchart of iTRAQ sample preparation and prediction of SnRK1.1 targets.Figure S4. The recombinant proteins of SnRK1.1 substrates and the phosphorylation pattern of IP-SnRK1.1 under submergence.Figure S5. Immunoprecipitation of SnRK1 in Col-0 and inactive SnRK1K48M in cMYC-SnRK1.1K48M.Figure S6. The profile of two PTP1 transgene lines.Figure S7. Examination of the interactions between PTP1S7AS8A and PTP1S7DS8D with negative controls.Table S1. Quantification of phosphoproteins in Col-0 and SnRK1.1K48M under submergence.Table S2. Quantification of proteins in Col-0 and SnRK1.1K48M under submergence.Table S3. Phosphorylation peptides up-regulated in Col-0 under submergence.Table S4. Phosphorylation peptides down-regulated in Col-0 under submergence.Table S5. Differential phosphorylation of selected submergence- responsive genes in SnRK1.1K48M mutants.Table S6. Protein abundance of differential phosphorylation responsive genes in SnRK1.1K48M mutants.Table S7. Phosphorylation motifs identified in whole seedings of Arabidopsis under submergence.Table S8. Primer list.


The anti‐human immunoglobulin E (IgE) monoclonal antibody, omalizumab , is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre‐existing IgE levels, and frequent dosing (q2–4 weeks). A vaccine inducing anti‐IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide‐conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non‐human primates, and demonstrate the induction of anti‐peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels. The incidence of IgE‐mediated allergic conditions, including allergic asthma and rhinitis, has significantly increased over the last few decades, resulting in significant morbidity and mortality and associated costs Despite the availability of multiple treatment modalities, including pharmacotherapy to treat symptoms and immunotherapy to address underlying immunological defects, there remains a significant unmet medical need for new therapeutic approaches to treat and potentially modify allergic diseases Elevated levels of immunoglobulin E (IgE) are characteristic of allergic diseases Immunoglobulin E (IgE) has long been used as a diagnostic biomarker, and more recently it has become a therapeutic target. Omalizumab  is a humanized anti‐IgE monoclonal antibody that directly targets free IgE, preventing it from binding to FcϵRI. Omalizumab was originally approved for the treatment of moderate to severe allergic asthma in patients who are not well controlled by inhaled corticosteroids In theory, an IgE targeting vaccine could provide clinical benefits similar to omalizumab except with less frequent dosing and relatively lower cost of goods A number of IgE peptide antigens were rationally designed, using structural information for IgE and its interaction with the high affinity FcϵRI receptor such that the anti‐IgE antibodies induced by the vaccine will bind free IgE, but not interact with IgE bound to cell surface receptors, thus avoid triggering the release of inflammatory mediators through IgE receptor cross linking Through extensive screening in mice two peptides were selected for further development. Screening involved immunization of mice with peptides conjugated to a virus‐like particle (VLP) that is derived from Qβ bacteriophage (Qb‐VLP) then ex vivo assessment of anti‐sera by ELISA for high titers of antibodies recognizing human IgE and in vivo functional assessment of rate of clearance of human IgE after bolus administration to mice. Selected peptides were also screened to ensure that induced antibodies do not induce degranulation of FcϵRI receptor‐expressing cells through binding to receptor bound IgE and that they do not induce a T cell responses. The two selected peptides, labeled peptide P and peptide Y, are derived from different loops of the Cϵ3 domain of IgE. Peptide Y corresponds to the epitope recognized by omalizumab while peptide P targets a different loop on IgE Cϵ3 Macacafascicularis) was selected since there is a ∼85% sequence homology between human and cynomolgus IgE constant region with a 100% homology for the P peptide and only single amino acid difference for the Y peptide In the current study, we evaluated the anti‐IgE vaccine in non‐human primates (NHP) for immunogenicity and function (lowering of circulating IgE). Cynomolgus monkey . Cells were lysed by homogenization in the presence of Triton X‐100 . Cell debris was removed by centrifugation. Qb‐VLP was purified using a series of chromatography steps: Fractogel TMAE , Ceramic Hydroxyapatite Type II , Phenyl Sepharose , and Sepharose CL‐4B . After the final column, the pooled fractions were concentrated to 3 mg/mL using a Pall Ultrafiltration system equipped with a 100 kD Pellicon 2 Biomax V‐screen PES cartridge. Samples were frozen at −80°C until further use.Qb‐VLP was made by Pfizer using Peptides P and Y were sourced from Chinese Peptide Company . The sequence of peptide P is ADSNPRGVSAYLSRPSPGGC and the sequence of peptide Y is QCRVTHPHLPRALMRS. Cysteines were added to each peptide sequence to allow conjugation through the sulfhydryl group of the added cysteine using the bifunctional linker, Succinimidyl‐6‐[ß‐maleimidopropionamido]hexanoate .Qb‐VLP conjugates were prepared by a two‐step process which first involved activating the Qb‐VLP with SMPH and secondly, conjugating with peptide. A 10× molar excess (for peptide P) or 4.25× molar excess (for peptide Y) SMPH in DMSO (Sigma–Aldrich) was added to 100 mg of Qb‐VLP at 3 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, pH 7.2. Activation proceeded at 15°C for 5 h with continuous mixing. After 5 h, the activated Qb‐VLP was purified and buffer exchanged into 100 mM sodium phosphate, 300 mM NaCl, pH 6.8 by ultrafiltration/diafiltration (UF/DF) using a Sartorius Slice UF system installed with a Biomax 300 kD membrane. The purified activated Qb‐VLP was diluted to 1 mg/mL with 100 mM sodium phosphate, 300 mM NaCl, pH 6.8, and peptide was added at a 7× molar excess. Conjugation proceeded at 15°C for 1.5 h with continuous mixing. After 1.5 h, the conjugated Qb‐VLP was purified and buffer exchanged into 100 mM sodium phosphate, 200 mM NaCl, pH 7.2 by UF/DF using a Sartorius Slice UF system installed with a Biomax 300 kD membrane. Sucrose at 140 mg/mL and PS20 at 0.2 mg/mL were added to the purified peptide‐Qb‐VLP conjugates. The concentration was adjusted to 2.5 mg/mL and the conjugates were frozen at −80°C until further use.3+.Aluminum hydroxide was obtained in the form of Alhydrogel “85”  and is hereafter referred to as alum, with doses indicating amount of AlCpG ODN (CpG) (B‐Class with sequence of 5′ TCG TCG TTT TTC GGT GCT TTT 3′) was synthesized by Avecia  with a nuclease‐resistant phosphorothioate backbone) as described previously n = 6 to 8/group) aged between 2 and 5 years were used in the studies. Circulating IgE titer in study animals ranged from 10 (lower limit of quantification for the assay) to 3141 U/mL at study start. Animals were randomized into groups with group Geomean titers (GMT) of circulating IgE ranging from 52 to 126 U/mL. Monkeys were immunized as indicated on figure legends with P peptide and/or Y peptide conjugated to Qb‐VLP  with CpG and/or alum. Doses of antigen and adjuvants are defined in figure legends. All vaccine formulations were made up to a total volume of 1.0 mL with PBS and administered by intramuscular (IM) injection in the left quadriceps muscle. Animals were bled at pre‐determined intervals  by saphenous vein puncture and plasma/serum used for quantitation of antibody specific to the peptides, whole IgE, or the C3C4 portion of human IgE. In some studies, plasma/serum was also used for quantification of total IgE titer. All procedures performed on animals in this study were in accordance with regulations and guidelines reviewed and approved by the Pfizer Institutional Animal Care and Use Committee and were conducted in facilities fully accredited by AAALAC International.Cynomolgus monkeys   using plasma or serum samples from individual animals. MaxiSorp 384‐well plates  coated (12 μL/mL) with whole human IgE , Qb‐VLP (3 μg/mL), Y or P peptides conjugated to KLH  were used for the detection of anti‐IgE, anti‐Qb, anti‐Y, and anti‐P titers, respectively. KLH was used for conjugation of peptides used in the ELISA in order to eliminate detection of any anti‐carrier antibodies. Reacti‐Bind™ Streptavidin HBC Clear 384‐Well Plates (Fisher Scientific) coated (12 μL/mL) with biotinylated human C3C4  were used to detect anti‐C3C4 titers. Selected samples were similarly tested on plates coated with cynomolgus C2C4 to compare anti‐human and anti‐cynomolgus antibody responses. Coated plates were incubated overnight at 4°C, except for plates coated with human C3C4 or cynomolgus C2C4 which were incubated for 1 h at room temperature. Following incubation, plates were aspirated and washed with PBS (for C3C4 or peptides) or PBS containing 0.05% Tween 20 (for whole IgE or Qb) and blocked 10% bovine calf serum  for 1 h at room temperature. Samples were serially diluted in dilution buffer (10% BCS in DPBS) and added to the blocked and washed plates (12 μL/well). Plates were incubated at room temperature for 1 h on a shaker. The plates were washed again with PBS‐0.05% Tween 20 and then incubated with goat anti‐human IgG‐horse radish peroxidase (HRP)  diluted with dilution buffer (10% BCS in DPBS) for 1 h at room temperature. The plates were then washed again and incubated with Tetramethylbenzidine (TMB) substrate  in the dark for 30 min at room temperature. The reaction was stopped by addition of 25 μL/well of 4N sulfuric acid (Fisher Scientific) and read at 450 nm using automated plate reader. Titers were defined as the highest serum dilution that resulted in an absorbance value (OD 450) two times greater than that of the diluent control. Group values were expressed as GMT ± 95% confidence interval (CI).Except in cases where anti‐IgE antibodies were detected with various coating reagents  for comparison purposes, C3C4 was selected for routine detection of anti‐IgE since it gave similar titers to those when plates were coated with whole human IgE.The levels of circulating IgE in individual plasma or serum samples were quantified using the Monkey IgE ELISA kit  as per manufacturer's instructions. The sample OD was determined at wavelength 450 nm with OD subtraction at 630 nm using a SPECTRAmax Plus Microplate Spectrophotometer. The pre‐existing endogenous IgE levels were measured in all animals at start of vaccination and the reduction in serum IgE is presented for individual animals as percent change in circulating IgE from baseline.50) was extrapolated for each sample tested. Using the competition ELISA whereby IC50 was determined at equilibrium, the IC50 value equates to Kd Antibody avidity was determined by competition ELISA as previously described Kd value was defined as amount of inhibitor required to achieve 50% inhibition of binding of anti‐IgE antibody present in plasma/serum to human IgE.Antibody specificity was determined using the same method as for avidity with the exception of using additional inhibitors; i.e., human IgG, IgM, IgA (Southern Biotech), and C3C4. For this assay, an equal volume of serially diluted inhibitor and diluted serum/plasma or the diluent (10% BCS in DPBS) were added directly to MaxiSorp 384‐well plates coated with human IgE . The concentration ranges of the inhibitors were from 2.5 to 158 nM. Omalizumab  was used as a positive control and the concentration ranges of the inhibitors for Omalizumab were from 0.2 to 13.1 nM. The t‐test and between three or more groups by one‐way or two‐way ANOVA followed by post hoc analysis using either Dunn's or Tukey's multiple comparison tests. Differences were considered to be not significant with p > 0.05.Data were analyzed using GraphPad Prism . Statistical significance of the difference between two groups was calculated by Student's 2‐tailed R2 = 0.98 (data not shown).Y‐Qb adjuvanted with alum and CpG was tested at different doses in cynomolgus monkeys for immunogenicity and the ability to decrease circulating IgE levels. Using plates coated with human C3C4, it was found that a small number of monkeys (6%) had a low level of pre‐existing anti‐IgE (titers between 100 and 500), however, these pre‐existing titers did not appear to interfere with vaccine immunogenicity and all animals showed an increase in anti‐IgE titers post vaccination relative to baseline  was tested and there was no antigen dose effect on the quality of the antibody response over this 20‐fold antigen dose range Fig. C.p < 0.05, Fig. As shown above, it was not possible to increase anti‐IgE antibody titers with antigen dose. Earlier studies in mice had shown the combination of Y‐Qb and P‐Qb to be superior to Y‐Qb alone for anti‐IgE antibody titer Plasma from animals dosed with Y‐Qb/P‐Qb combination using alum and CpG as adjuvants were tested for antibodies specific to Y and P peptides, C3C4, whole IgE, or the Qb VLP carrier. Animals had pre‐existing antibody titer ≤ 0.25 K GMT against the Y and P peptides, C3C4, whole IgE, or the Qb VLP and titers developed with similar kinetics post immunization. Although these assays are not strictly comparable due to the use of different coating reagents for the sandwich ELISA, similar antibody titers were detected with IgE and C3C4 . Therefore, C3C4 protein was used as coating reagent for all other ELISA data reported in this manuscript. Anti‐Y antibody titer (GMT 77K) was also comparable to that for anti‐whole IgE whereas the anti‐P titers were higher (GMT 262K). As expected, high antibody titers developed against the carrier Qb VLP (GMT 633K)  to 10 or 100 μg . Anti‐IgE antibodies induced in this study were specific for human IgE and did not cross react with human IgG, IgA, or IgM  calculated by ELISA for vaccine‐induced antibody against whole human IgE was 36 ± 11.6 nM whereas that for omalizumab was 0.6 nM using the same assay.In a separate study, lower doses of Y‐Qb + P‐Qb  were tested with alum (250 μg) and CpG (500 μg). Following dosing at 0, 4, and 12 weeks, there was no difference in antibody titer except at 2 weeks after the last dose where 1 µg was inferior  , even when analysis was done on a subset of animals with the highest anti‐IgE titers .It is possible that the presence of endogenous anti‐IgE antibodies may in theory impact the detection of serum IgE levels using the commercial sandwich ELISA kit employed in this study. However, since the vaccine‐induced responses are directed against specific epitopes within the Cϵ3 domain and the diagnostic anti‐IgE reagent is polyclonal, the impact should be minimal. This is confirmed by the fact that in some animals no IgE lowering is detected despite the presence of high levels of anti‐IgE antibodies while in other animals different levels of IgE lowering despite similar magnitude of anti‐IgE titer. There was no correlation between reduction in circulating IgE and anti‐IgE antibody titer or avidity (At a low antigen dose (20 μg of Y‐Qb + P‐Qb combination), CpG inclusion with alum in the adjuvant formulation significantly enhanced the anti‐IgE antibody response compared to alum alone. However, this benefit of CpG was not statistically significant when a high antigen dose (900 μg) was used  Peptide screening studies in mice had identified murine homologues of Y‐Qb (mY‐Qb) and P‐Qb (mP‐Qb) as the best peptide conjugates for induction of strong antibody responses that cross‐react with the intact IgE molecule. In mice, both mY‐Qb and mP‐Qb induced similar anti‐IgE responses with the combination being superior to either peptide conjugate alone. However, mY‐Qb was superior to mP‐Qb in lowering serum IgE There was a mixed antigen dose effect on immunogenicity in NHP. For anti‐IgE antibody titers, the antigen dose response was absent or small. For example, with Y‐Qb alone, there was no evidence of an antigen dose response from 45 to 900 µg for titers of anti‐IgE Ab. With Y‐Qb and P‐Qb combined, Ab responses were weaker with very low doses of the combination vaccine  compared to higher doses (20 or 200 µg) for at least some time points. An antigen dose effect was also not evident for antibody avidity. Similarly, in mice a weak dose response was seen with mY‐Qb/mP‐Qb combination at lower antigen dose levels All vaccine formulations were adjuvanted since earlier studies in mice and dogs showed very poor immunogenicity for antigen conjugates alone . Most groups received both alum and CpG, but results with alum alone were not inferior to those with alum/CpG except at the lowest antigen dose, indicating a lack of CpG effect once a certain dose of antigen is used. It has been well‐established with a wide variety of antigens, but not including Qb‐VLP conjugates, that alum/CpG significantly enhances antibody responses over alum alone, including in humans studies Some animals had pre‐existing anti‐IgE antibodies, which did not appear to impact the response to the vaccine. The presence of auto anti‐IgE antibodies is well‐established in humans, and have been shown to cause modest (<50%) inhibition of in vitro IgE binding to high affinity receptors using a FcϵRI‐expressing cell‐line, as well as to low affinity CD23 receptors on B cells, suggesting possible anti‐allergic and pro‐allergic effects, respectively The vaccine‐induced anti‐IgE antibodies resulted in significant lowering of serum IgE levels in many of the animals, with some animals achieving as much as 80% lower IgE compared to baseline. Despite the lack of antigen dose effect on antibody titer and avidity, there was a clear trend for better functional responses (IgE lowering in blood) with higher antigen doses, suggesting that the assays for titer and avidity are not adequate predictors of anti‐IgE vaccine function. However, despite an increase in the overall antibody titer, addition of P‐Qb with Y‐Qb did not appear to improve the functionality of the antibody over what was seen with Y‐Qb alone at same dose. This may be reflective of the poor quality of anti‐P antibodies; likely due to conformational restrictions of the epitopes within the P peptide. Various studies have illustrated the benefits associated with introducing conformational constraints such as disulphide constrained loops to enhance affinity of peptide vaccines In clinical studies, adequate dosing of omalizumab has been shown to reduce serum free IgE levels by more than 95% in the majority of patients Predicted free IgE levels in asthmatic patients treated with omalizumab were correlated with reported clinical outcomes and a target level of 14 ng/mL was selected to achieve desired clinical benefit, with clear loss of clinical control above ∼30 ng/mL Although it was beyond the scope of the current study to evaluate therapeutic efficacy of the vaccine‐induced antibodies in monkeys, this was evaluated using mouse and dog mimetics. With mouse prophylactic and therapeutic allergy models and in dog hypersensitive to house dust mites, mimetic vaccines to the human anti‐IgE vaccine showed clear symptomatic benefit despite serum IgE levels being reduced less than 90% Omalizumab binds with high affinity to free IgE, preventing its binding to the FcϵRI receptor and while free IgE is drastically reduced, total IgE is greatly increased. Such an increase in total IgE was not detected with the vaccine described here in and pre‐clinical data in mice suggest that the anti‐IgE antibodies do not prevent IgE from binding to its receptor, but rather there is an increased clearance rate of IgE with reductions of both free and total IgE In summary, a novel peptide conjugate anti‐IgE vaccine was tested in NHP and elicited anti‐IgE antibodies that, at least in some animals, could bring about substantial IgE lowering after 3 or 4 vaccine doses. Although the degree of IgE lowering in NHP is less than that seen with omalizumab in humans, the differences in species and potential mechanism of action make it hard to predict how this would translate into humans. Therefore, a decision was made to advance this anti‐IgE vaccine into human clinical testing.None declared.


Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish ( Tyrosine kinases with Immunoglobulin and Epidermal growth factor homology domains (Tie) receptors, Tie1 and Tie2, are known to play important roles in vascular growth, remodeling, and maintenance, as knockout of either Tie1 or Tie2 results in embryonic lethality due to vascular defects. Tie2 knockout mice die midway through gestation by embryonic day (E) 10.5 with vessel sprouting defects, whereas mice lacking Tie1 die between E13.5–15.5 or perinatally with severe vascular integrity defects, including hemorrhage and edema YQSGSGE-amide & C-Ahx –LHRRR[pT]F[pT]YQSGSGE-amide. Rabbit polyclonal Tie1 C-tail antibody , mouse monoclonal phospho-Tyrosine antibody , and rabbit polyclonal Tie2 C-tail antibody  were from Santa Cruz Biotech . Extracellular Tie2 mouse monoclonal antibody (mAb 33) was from Amgen . Rabbit polyclonal phospho-Akt substrate (#10001), phospho-Akt (Ser473) (#9271), total Akt (#9272), total p44/42 MAPK , Rac 1,2,3 (#2465), phospho-PAK1 (S144)/ PAK2 (S141) (#2606), PAK1 (#2602), and mouse P-p44/42 (#9106) were from Cell Signaling Technology . Rat alpha-Tubulin (#MCA77G) antibody was from AbD Serotec . Rho/Rac/Cdc42 activator (#CN04), recombinant His-Rac1 protein (#RC01) and ROCK inhibitor Y–27632 (#CN06) were from Cytoskeleton Inc . PAK inhibitor IPA 3 (#3622) was from Tocris Biosciences . Complete® Protease inhibitor and PhosStop® phosphatase inhibitor were from Roche . HUVECs were isolated in lab from umbilical cords donated to the Duke University Cord Blood Bank as previously described [Rabbit polyclonal phospho-Tie1-T794 antibody was generated by 21escribed . Human eescribed . Matrige® protease inhibitor tablets, PhosSTOP® phosphatase inhibitor) and incubated with 1mM ATP, kinase buffer  and endothelial cell lysate for 1hr at RT with mixing. The GST-beads were then washed twice more with lysis buffer before the proteins were eluted into Laemmli buffer to be resolved by SDS-PAGE and western blot.0.5–1.0μg of GST-Tie kinase fusion protein was precipitated from SF9 insect cell lysates using 30ul Glutatione-sepharose beads . Purified proteins were washed in Triton lysis buffer (20mM Tris (pH 8.0), 137mM NaCl, 2mM EDTA (pH 8.0), 10% v/v Glycerol, 1% v/v Triton-X, CompleteDanio rerio) embryos were raised and maintained as described [tie1 (5’-CATGTCTACTTACAGATCCAGATTG–3’) was reported previously [All zebrafish embryo studies were approved by the Duke University Institutional Animal Care and Use Committee. Zebrafish (escribed . 24 houreviously  and was -T794A mRNAs were cloned into the pCS2 vector and transcribed in vitro using the SP6 Message Machine kit (Ambion). Numbers of embryos per group were as follows: Uninjected, n = 300; Control MO, n = 62; Tie1-MO (MO), n = 133; MO+Tie1-WT, n = 105; MO+ Tie1-T794A, n = 79; Tie1-T794+WT, n = 27; Tie1-WT, n = 275; Tie1-T794A, n = 60.1nl of diluted MO (5 ng) and/or RNA (50 or 100 pg) was injected into Fli1:EGFP zebrafish embryos at the 1- to 2-cell stage. Injected embryos in each group were blinded by a member of the Katsanis lab not involved with the project and classified at 48 hpf (by C. Golzio) as normal or mutant based on relative vasculogenic defects compared with age-matched controls from the same clutch. Mutant embryos were characterized by a minimum of four missing intersegmental vessels (ISV) and/or the absence of the dorsal longitudinal anastomotic vessel (DLAV). For RNA rescue experiments, the murine Tie1-wild type and Tie18 pfu/ml +/- AdmyrAkt and incubated overnight. Ligand stimulation experiments were starved for 3h prior to incubation with ligand as indicated. All CN04-mediated Rac1 activation experiments were starved overnight followed by a 30 min pre-treatment with inhibitors and/or 3h CN04 (5μg/ml) incubation. Cells were then washed once with PBS and lysed in Triton lysis buffer containing protease and phosphatase inhibitors. Lysates and immunoprecipitates were resolved by SDS-PAGE and analyzed by western blot.HUVECs were infected with GFP or Tie1 adenoviruses at ~3x108 pfu/ml. Cells were then plated on Matrigel (10mg/ml) at 6,000 cells/well of a 96-well plate. Remaining cells were lysed and analyzed by western blot to assure equal adenoviral Tie1 expression. Brightfield images were captured using an inverted epi-fluorescent (6x objective) microscope 6 hours after plating and quantified using ImageJ-Angiogenesis Analyzer macro. Fluorescent images were taken by fixing tubes in 4% PFA, permeabilizing in 0.5% Triton-X, and staining with 70nM Rhodamine-Phalloidin.HUVECs were infected overnight with GFP or Tie1 adenoviruses at ~3x10Tie1 antibody  was covalently conjugated to Protein-A agarose beads using a Dimethylpimelimidate (DMP)-crosslinking protocol provided by the Duke Proteomics Core Facility. In brief, antibody was coupled to agarose beads by mixing for several hours. Beads were then washed 3x in a 0.2M sodium borate buffer (pH 9.0) to remove uncoupled antibody. Antibody was cross-linked using freshly made 20mM DMP in 0.2M sodium borate (pH 9.0) for 40 minutes at room temperature. Beads were then washed in 0.2M ethanolamine to quench the DMP reaction, then subjected to an acid wash (0.58% v/v acetic acid + 150 mM NaCl) 3x to remove uncoupled antibody. Coupled beads were then validated for efficiency and specificity by Coomasie stain and Western blot, respectively.Immortalized endothelial cells (ECRFs) constitutively expressing a human Tie1 shRNA were infected with adenoviruses expressing mouse Tie1-WT or T794A +/- AdmyrAkt for 24 hours then lysed in NP–40 lysis buffer. Tie1 was immunoprecipitated on cross-linked beads and washed thoroughly with NP–40 lysis buffer. Samples were then washed 3x in 50mM Ammonium Bicarbonate and mixed with 0.2% Rapigest SF Surfactant (Waters Corporation) and delivered to the Duke Proteomics Core Facility for in solution digestion and proteomic analysis of associated proteins. Unique peptides were identified using the SwissProt database. Peptides that bound to the IgG-beads control or known to non-specifically bind to the Protein A/G agarose beads were discarded as non-specific. Proteins identified by 2 or more unique peptides were considered to be specific binders.HUVECs were infected with GFP or Tie1 adenoviruses and lysed in NP–40 lysis buffer  containing protease and phosphatase inhibitors. Lysates were incubated on a rotator at 4°C with 1ug of recombinant His-Rac1 for 6 hours. Aliquots of whole cell lysate were collected at this point to analyze by Western blot. The remaining lysate was mixed with Tie1 antibody overnight. Protein-A Agarose beads were added for 1 hour the following morning. Proteins precipitated on the beads were then washed 3 times with NP–40 lysis buffer and mixed with 1× Laemmli running buffer containing 5% β-mercaptoethanol, boiled for 5 minutes, and resolved by SDS-PAGE.HUVECs were infected with the indicated viruses overnight then plate at 10,000 cells/well of a 6-well plate. Every 24 hours following plating, separate groups of cells were fixed in cold methanol for 5 minutes, then allowed to dry before staining with 50% hematoxylin. Four random images were captured per well and number of nuclei were counted. Cell numbers were normalized to the mean number of cells 24 hours after plating.Nearly-confluent HUVECs were left uninfected or were infected with the indicated adenovirus. 24 hours after infection, the media was changed to either full-serum media or serum- and growth factor-free starvation media. 48 hours later, the cells were washed once with PBS to remove dead and non-adherent cells, fixed in methanol, hematoxylin stained, and random fields were imaged with a 6× objective. Nuclei were counted and normalized to the average number of cells in the full-serum media group for each virus condition to account for any proliferation that may have taken place.6) were then incubated with the HUVECs for 30 minutes at 37°C. Non-adhered cells were washed away. Brightfield images (4x objective) were captured and adherent U937s were counted.Confluent HUVECs were infected for 24 hours with the indicated adenovirus, then treated with or without TNFα (1ng/ml) overnight. U937 leukocytes  coated with gelatin (0.1%). Full-serum growth media was added to the lower chamber and cells were allowed to migrate for 6 hours. Unmigrated cells were then removed from the top of the membrane using a cotton swab. Migrated cells were fixed in methanol for 15 minutes and incubated with a DAPI solution (300nM in PBS) for 5 minutes. Random fields were chosen and counted.All western blots are representative of at least 3 repeat experiments. Quantification of tube formation data was the result of 11 independent experiments, each normalized to uninfected controls for comparison. Zebrafish data are expressed as a percentage of the total number of embryos counted per group. Significance was determined by a Chi squared test. All other results are expressed as the mean ± the standard error of the mean (SEM). Analysis of variance was used to compare means of different groups and Student’s t-tests were used to determine individual differences. In all instances, P<0.05 was considered statistically significant.in vitro kinase reactions using a fusion protein comprised of the Tie1 JM and kinase domains fused to GST (GST-Tie1) incubated with endothelial cell lysates that had been infected with either a control, empty adenovirus (AdEmpty) or constitutively active, myristoylated Akt adenovirus (AdmyrAkt). Western blotting with a phospho-Akt-substrate antibody, which detects the RXRXXpS/T sequence on many Akt substrates, demonstrated that Tie1 was phosphorylated after incubation with lysates from AdmyrAkt infected cells but not AdEmpty infected cells  lies within a high-probability Akt consensus phosphorylation sequence . To tested cells . To valied cells .in vitro transcribed capped mRNA encoding the WT mouse Tie1 was co-injected together with Tie1 MO, which significantly rescued the morphant phenotype  . A validogenesis .in vitro. HUVECs infected with these adenoviruses were plated on Matrigel and allowed to form tubes for 6 hours before fixation, phalloidin staining, and imaging . Mass spectrometry revealed a number of Tie1-interacting proteins . Among tGiven our negative results with any physiological activators of Akt to induce T794 phosphorylation, the interaction of Tie1 with Rac1 raised the possibility of a feedback loop involving effector kinases downstream of the Rho GTPases, such as PAK1 and ROCK. Therefore, we assessed these serine/threonine kinases for their potential ability to phosphorylate Tie1-T794. Treatment of HUVECs with CN04, a Rho/Rac/Cdc42 activator that induces activation of both PAK1 and ROCK, resulted in readily detectable T794 phosphorylation, which was abrogated by expression of the T794A mutant . To detein vivo and Tie1 knockdown or overexpression in ECs in vitro have provided some functional insights, however these studies have provided little clarity regarding Tie1’s mechanism of action. For instance, Tie1 overexpression in vitro results in upregulation of inflammatory genes such as ICAM, VCAM, and VE-Cadherin, whereas silencing Tie1 in ECs decreases expression of these inflammatory genes and proteins. Accordingly, EC-specific conditional deletion of Tie1 limited vascular inflammation and the development of atherosclerosis in ApoE-/- mice [An essential role for Tie1 in embryonic vascular development was demonstrated by two separate groups 20 years ago. Recent studies in conditional Tie1 knockout mice -/- mice . More re-/- mice . DecreasTie1 is generally still classified as an orphan receptor, as it is unable to bind directly to any known ligands, including the angiopoietins, the ligands for Tie2. Saharinen et al demonstrated that Tie1 could be activated by Ang1, and receptor activation (tyrosine phosphorylation) was enhanced in the presence of Tie2, suggesting that Tie1 could serve as a co-receptor for Tie2 and possibly other receptors, such as integrins, which play a role in angiopoietin-Tie2 signaling –29. Crysin vivo in zebrafish and in matrigel suggests an important role for cell-matrix interactions, such as those mediated by integrins, although this remains to be proven. Taken together, however, our results suggest that Tie1 may not act as a canonical RTK, but perhaps more like the EGF receptor ErbB2, which does not bind ligand but is able to multimerize with and modulate the signaling of the other EGF receptors [To circumvent this problem, our group generated a CSF–1 receptor-Tie1 chimera and demonstrated that the Tie1 kinase could be ligand-activated and transduce cell survival signals through PI 3-kinase and Akt . Consisteceptors . In addiin vivo T794 phosphorylation, we cannot rule out the possibility that the T794A mutant affects vascular development in a phosphorylation-independent manner. It is unlikely that the T794A point mutation would adversely affect translation or proper folding of the Tie1 protein, as the same point mutant was readily expressed in vitro and behaved functionally similar to Tie1-WT in non-angiogenic contexts. However, it is possible that it could alter protein-protein interactions in a phosphorylation-independent manner. Nonetheless, our in vivo findings, together with complementary in vitro tube formation assays, Rac1/PAK1-mediated T794 phosphorylation studies, and Rac1’s known role in sprouting angiogenesis strongly suggest that the ability to modulate the phosphorylation state of T794 may be critical for proper angiogenesis and capillary morphogenesis. These data also suggest a role for Tie1 in modulation of the endothelial cytoskeleton, a role that has not been demonstrated previously.Using the zebrafish model, we were able to assess the biological importance of the putative Akt consensus phosphorylation site at Thr794. In support of the specificity of our approach, knockdown of Tie1 resulted in defects in angiogenesis and vascular integrity similar to those shown previously in mice , 33, 34 Ser/Thr phosphorylation has been demonstrated for a number of RTKs and has been shown most commonly to negatively regulate kinase activity and downstream signaling –14. SomeLike most small GTPases, Rac1 cycles between an active GTP-bound state and an inactive GDP-bound state. Basal Rac1 signaling generally mediates endothelial cell barrier protection, whereas Rac1 activation promotes intercellular attachments and regulation of actin and microtubule rearrangement during endothelial lumen formation . As within vivo, neither can we confirm Tie1 as an Akt substrate as defined by Manning and Cantley [Considering that we suspected T794 to be an Akt phosphorylation site, the lack of an effect of the T794A mutant on cell survival and proliferation was particularly surprising and raised the question as to whether another kinase might be involved. Although the RRRTFTY sequence in the Tie1 JM domain could be phosphorylated by overexpression of constitutively active Akt, we were unable to identify a physiological stimulus that induced Akt-mediated phosphorylation of T794, raising concerns about promiscuity following overexpression of constitutively active Akt . While o Cantley . While i Cantley . Accordi Cantley . Our resThe finding that T794 phosphorylation occurs downstream of Rac1 activation, considered together with the fact that both Rac1 and Tie1-T794 are required for angiogenesis, suggests the presence of a feedback loop involving Rac1 activation, PAK1-mediated Tie1 phosphorylation, Rac1-Tie1 association, and subsequent modulation of angiogenesis . WhetherS1 ARRIVE ChecklistCompleted ARRIVE Guidelines checklist.(DOCX)Click here for additional data file.S1 DatasetZIP file containing raw data from Tie1 proteomic interactions following overexpression of Tie1-WT or -T794A in endothelial cells infected with or without AdAkt.(ZIP)Click here for additional data file.S1 FigHUVECs were infected for 24h with adenoviruses encoding the indicated proteins then plated in 8μm Transwell filters coated with gelatin (0.1%) and allowed to migrate for 6h. Migrated cells were then fixed in methanol, stainted with DAPI and nuclei of migrated cells were then imaged by epiflourescence microscopy. Cells were counted in 3 random fields (4x magnification) using Image J. Data represents relative means ± SEM. * GFP vs TA, P<0.001.(TIF)Click here for additional data file.S2 FigHUVECs were left uninfected or infected with AdTie1-WT or -T794A for 24 hours, starved in serum-free medium for 3 hours, then stimulated for 15 minutes with Ang1 (500ng/ml). Tie1 was immunoprecipitated (IP) from cell lysates and western blotted as indicated. Tie1-WT and T794A bands are from the same blot and exposure.(TIF)Click here for additional data file.S3 FigHUVECs were uninfected or infected with AdTie1-WT or -T794A for 24 hours, starved in serum-free medium for 3 hours, then stimulated for 15 minutes with Ang1 (500ng/ml). Tie2 was immunoprecipitated (IP) from cell lysates and western blotted as indicated.(TIF)Click here for additional data file.S4 Fig6) were then incubated with the HUVECs for 30 min at 37°C, and non-adhered cells were washed away. Brightfield microscopy images were taken and the number of adherent U937 leukocytes was counted in 3 random 4x fields. Tie1 overexpression enhanced the inflammatory response to TNFα but in a T794-independent manner. Results are shown as means ± SEM. *, P<0.04.Confluent HUVECs were infected for 24h with the indicated proteins and then treated with or without TNFα (1ng/ml) overnight. U937 leukocytes (3x10(TIF)Click here for additional data file.S5 Figa. Uninfected or Adeno-infected HUVECs were left untreated or starved for 48 hours. Cells were fixed, stained with hematoxylin, and counted. Cell counts were normalized to the average number of cells per field in the untreated group for each virus group. b. HUVECs were infected for 24h with adenoviruses encoding the indicated proteins, and then sparsely plated 6-well plates. Separate groups of cells were fixed, stained, and counted every 24h after plating. Results are expressed as relative mean cell number ± SEM. Overexpression of Tie1 suppressed proliferation in a T794-independent manner. *, P<0.001.(TIF)Click here for additional data file.S6 FigHUVECs adenovirally overexpressing Tie1-WT were starved for 3h and stimulated for 15 minutes at 37°C with the following ligands: Ang1 (500ng/ml), Ang2 (500ng/ml), VEGF (25ng/ml). Tie1 was immunoprecipitated from whole cell lysates and both were western blotted as indicated. Bands from the AdmyrAkt-treated cells were from the same blot and exposure.(TIF)Click here for additional data file.S1 TableTie1 antibody was conjugated to beads and used to immunoprecipitate (IP) adenovirally overexpressed Tie1-WT or -T794A from endothelial cells that had also been infected with or without AdAkt. IPs were thoroughly washed and submitted to the Duke Proteomic core facility for mass spectrometric (LC-MS/MS) analysis.(TIF)Click here for additional data file.


P < 0.01). The amount of reciprocal Ia inhibition increased with fast movement speed conditioning (P < 0.01), but was unchanged by slow movement speed conditioning. These changes lasted up to 15 min in D1 inhibition and 5 min in reciprocal Ia inhibition after the training session. The control task did not induce changes in D1 inhibition and reciprocal Ia inhibition. The TMS conditioned inhibitory effects of presynaptic inhibitory pathways decreased following visuomotor tasks (P < 0.01). The size of test H-reflex was almost the same size throughout experiments. The results suggest that supraspinal descending inputs for controlling joint movement are responsible for changes in the spinal neural circuits, and that task movement speed is one of the critical factors for inducing plastic changes in reciprocal Ia inhibition.Previous studies have shown that spinal neural circuits are modulated by motor skill training. However, the effects of task movement speed on changes in spinal neural circuits have not been clarified. The aim of this research was to investigate whether spinal neural circuits were affected by task movement speed. Thirty-eight healthy subjects participated in this study. In experiment 1, the effects of task movement speed on the spinal neural circuits were examined. Eighteen subjects performed a visuomotor task involving ankle muscle slow (nine subjects) or fast (nine subjects) movement speed. Another nine subjects performed a non-visuomotor task (controls) in fast movement speed. The motor task training lasted for 20 min. The amounts of D1 inhibition and reciprocal Ia inhibition were measured using H-relfex condition-test paradigm and recorded before, and at 5, 15, and 30 min after the training session. In experiment 2, using transcranial magnetic stimulation (TMS), the effects of corticospinal descending inputs on the presynaptic inhibitory pathway were examined before and after performing either a visuomotor (eight subjects) or a control task (eight subjects). All measurements were taken under resting conditions. The amount of D1 inhibition increased after the visuomotor task irrespective of movement speed ( Plastic changes in cortical areas induced by motor skill training have been investigated, and results suggest they are related to the acquisition of motor skills ] participated in this study after providing written informed consent. Baseline characteristics of participants are shown in Table Subjects were seated in an armchair with the examined leg semi-flexed at the hip (120°) and the knee (120°), and plantar-flexed at the ankle (110°). The right lower leg was secured with an ankle-foot orthosis brace. All experimental measurements were taken while in a resting condition.Electromyographic (EMG) activity was recorded with bipolar surface electrodes  placed on the right soleus (SOL) and tibialis anterior (TA) muscle belly. Raw EMG signals were amplified at 1000 times and band-pass filtered between 5 and 3000 Hz, using an amplifier . The EMG signals were digitized by an analog/digital (A/D) converter with a sampling rate of 10 kHz  and stored on a computer for subsequent analyses. The recording period was 200 ms including the pre-stimulus period of 50 ms.SOL H-reflex and M-wave were evoked by stimulating the posterior tibial nerve through a monopolar stimulating electrode (1 ms rectangular pulse) using a constant current isolator  coupled with an electrical stimulator . An anode was placed above the patella, and a ball cathode was placed at the popliteal fossa. The H-reflex and M-wave responses were measured as peak-to-peak amplitudes of the non-rectified reflex. At the beginning of the experiment, the maximum amplitudes of H-reflex (Hmax) and M-wave (Mmax) were recorded in all participants. As the sensitivity of the H-reflex to facilitatory or inhibitory conditioning inputs was changed by the test H-reflex size, the size of the control SOL H-reflex was adjusted to 20–30% of Mmax in all conditions  suppression of the SOL H-reflexes, by conditioning stimuli to the common peroneal nerve (CPN) that innervates the TA muscle. This long-lasting SOL H-reflex suppression, which is called D1 inhibition  suppression of the SOL H-reflex by a conditioning stimulus to the CPN. This H-reflex suppression is believed to reflect reciprocal Ia inhibition  through a double-cone coil connected to a magnetic stimulator . The coil was placed on the scalp to induce a posterior-anterior current flow in the left M1. An optimal stimulus position for evoking motor evoked potentials (MEPs) in the right SOL muscle with a weak contraction was assessed by moving a coil around the leg motor area and was determined as the site where TMS at a slightly suprathreshold intensity regularly produced the largest MEPs. This position was marked with a pen on a swimming cap worn by the subject. Active MT was defined as the minimal stimulus intensity required to induce MEPs of at least 200 μV in three of five trials in the SOL muscle. While measuring the active MT, special care was given to maintain constant EMG activity levels of the SOL muscle . The intensity of conditioning TMS was set to 80–90% of the active MT, so that they had minor facilitation effects on the test H-reflex in a resting condition. To examine the effect of corticospinal descending inputs on the presynaptic inhibitory pathway, we compared the amount of D1 inhibition in the absence and presence of TMS. The time interval between TMS and CPN stimulation was set at 5 ms. Moreover, to study TMS conditioning effects on the test H-reflex, the amplitude of TMS conditioning H-reflex was evaluated at C-T intervals of 26 ms. For measurement of short latency facilitation effects, the intervals between conditioned TMS stimulation and H-reflex stimulation was set at −3, −2, and −1 ms. The C-T interval that produced the first facilitation effects on the H-reflex was regarded as a suitable C-T interval, and was used throughout the experiment. Negative C-T intervals indicated that the conditioned stimulus was applied after the test stimulation. Conditioned and unconditioned H-reflexes were randomly evoked at 0.2 Hz.A custom made PC program  was used to set up a visuomotor task. The subjects were allowed free movement of the ankle joint when performing the motor task. For a visuomotor task, subjects repeatedly moved their ankle between target lines according to auditory beep sounds delivered at a frequency of 1 Hz or 3 Hz Figure . In thisn = 9), fast speed group (n = 9), and control group (n = 9).Twenty-seven subjects participated in Experiment 1. They were randomly allocated to three different groups: slow speed group  for 20 min. For the non-visuomotor task, the control group subjects repeatedly moved their ankle according to auditory beep sounds delivered at a frequency of 3 Hz (fast movement speed) without visual feedback of ankle movement. In this task, the subjects were not required precise control of joint movement. Other task procedures used the same visuomotor task.We measured the amount of D1 inhibition and reciprocal Ia inhibition before (pre), 5 min after (post 5), 15 min after (post 15) and 30 min after (post 30) the task sessions. We also tested the ratio of Hmax vs. Mmax (Hmax/Mmax), which was used as an indicator of motor neuron pool excitability, before and immediately after the task session. Hmax and Mmax were evoked every 3 s and calculated from the average of five Hmax and five Mmax values.Sixteen subjects participated in Experiment 2. Five of the sixteen subjects also took part in Experiment 1, and the time between each experiment was at least 2 months. In Experiment 2, eight subjects performed the visuomotor task (visuomotor group) in the fast movement speed condition (3 Hz), and eight subjects performed the control task (non-visuomotor group). Task procedures were the same for the Experiment 1 protocol.The amount of D1 inhibition, the amount of TMS conditioned D1 inhibition, the amplitude of TMS conditioned test H-reflex, the amplitude of TMS conditioned H-reflex amplitude at short facilitation phase, and Hmax/Mmax were measured before and after the task sessions.t-test or one-way analysis of variance (ANOVA). The performance data compared among the task sessions used the one-way repeated measure of ANOVA for each group. Test H-reflex size was compared using two-way repeated measures of ANOVA with the factors “time” and “group.” In the Experiment 1 protocol, two-way repeated measures of ANOVA with the factors “time” and “group” were used to evaluate the effects of movement speed of the visuomotor task on the D1 inhibition and reciprocal Ia inhibition. In addition, the amounts of D1 inhibition and reciprocal Ia inhibition were compared using one-way repeated measure of ANOVA in the control group. In the Experiment 2 protocol, the amount of D1 inhibition, the amount of TMS conditioned D1 inhibition, the difference in the amount of D1 inhibition in the absence and presence of TMS, the amplitude of TMS conditioned test H-reflex, and the amplitude of TMS conditioned H-reflex at short latency facilitation phase were analyzed by two-way repeated measures of ANOVA with factors “time” and “group.” For multiple comparisons, if significant effects were detected, the Bonferroni post hoc test was used. Mauchley’s test was used to examine for sphericity. The Greenhouse-Geisser correction was used for non-spherical data. The amount of D1 inhibition was compared with the amount of TMS conditioned D1 inhibition within the group, using the paired t-test. The amplitude of TMS conditioned H-reflex at short latency facilitation phase was also compared with the unconditioned H-reflex, using the one-sample paired t-test. Moreover, in both experiments, the differences in the Hmax/Mmax and SOL Mmax were compared between before and after the task within the group, using the paired t-test. P values of < 0.05 were considered significant in all statistical analyses. Data were analyzed using SPSS version 22 software . The data values are presented as the means ± standard error of the mean (SEM).The baseline characteristics of groups  were analyzed using the unpaired F2,24 = 0.32, P = 0.73 in the Experiment 1 group, t14 = 1.41, P = 0.18 in the Experiment 2 group; SOL Mmax: F2,24 = 0.13, P = 0.87 in the Experiment 1 group, t14 = 0.69, P = 0.50 in the Experiment 2 group; Hmax/Mmax: F2,24 = 0.36, P = 0.69 in the Experiment 1 group, t14 = 0.06, P = 0.94 in the Experiment 2 group; active MT: t14 = 0.41, P = 0.68; Stimulation intensity: t14 = 0.83, P = 0.42).Baseline characteristics of subjects among groups were well-matched in the Experiment 1 and the Experiment 2 groups . In post hoc tests, a significant reduction in the mean errors was observed between the first block and five (P = 0.04) and sixth (P = 0.01) blocks in the slow speed group. Similarly, a significant reduction in the mean errors was observed between the first and fourth blocks (P = 0.01), between the first and fifth (P = 0.02) and sixth (P < 0.01) blocks in the fast-speed groups. These results indicated that the task performance was certainly improved across blocks in both groups. However, there were no significant differences in the mean errors across the blocks in the control group .The mean times of one cycle of ankle movement are shown in Table F3,72 = 0.95, P = 0.41; reciprocal Ia inhibition: F3,72 = 1.48, P = 0.23) and “group”  on the test H-reflex amplitude. Likewise, there was also no significant “time” × “group” interaction .The mean amplitudes of the test H-reflex (% of Mmax) for all conditions are summarized in Table F2.06, 32.99 = 14.84, P < 0.01), but not of the “group” . There was no significant “time” × “group” interaction . Post hoc analysis of the “time” factor indicated that compared to pre, the amount of D1 inhibition was significantly increased in post 5 min (P < 0.01) and in post 15 min (P < 0.01). A two-way repeated measures of ANOVA for reciprocal Ia inhibition showed a significant effect of the “time” , but not of the “group” . Moreover, there were also significant “time” × “group” interactions . Post hoc analysis indicated that, in the fast-speed group, the amount of reciprocal inhibition was significantly increased in post 5 min—there were significant difference between pre and post 5 min (P = 0.01), post 5 min and post 15 min (P < 0.01), and post 5 min and post 30 min (P = 0.01). The amount of reciprocal Ia inhibition was also significantly different between slow and fast speed groups in the post 5 min time period (P = 0.03).The effects of the movement speed of a visuomotor task on the D1 inhibition and reciprocal Ia inhibition are shown in Figure t8 = 1.22, P = 0.26 in slow-speed group; t8 = 0.40, P = 0.69 in fast-speed group). The mean Hmax/Mmax after the motor tasks was 0.51 ± 0.07 in the slow-speed group and 0.58 ± 0.07 in the fast-speed group. There were also no significant differences in the Hmax/Mmax between pre and post in both groups .The mean SOL Mmax after the motor task was 13.86 ± 1.18 mV in the slow-speed group and 14.84 ± 2.09 mV in the fast-speed group. There were no significant differences in the SOL Mmax between pre and post in both groups  and reciprocal Ia inhibition . The mean SOL Mmax after the control task was 14.02 ± 1.33 mV and the mean Hmax/Mmax after the control task was 0.55 ± 0.05. There were also no significant changes in the SOL Mmax , and Hmax/Mmax  following the control task.The effects of the control task on the D1 inhibition and reciprocal Ia inhibition are shown in Figure F1,14 = 0.39, P = 0.54; TMS conditioned D1 inhibition: F1,14 = 4.07, P = 0.06; TMS conditioned H-reflex at short latency facilitation phase: F1,14 = 3.85, P = 0.07) and “group”  on the test H-reflex amplitude. There was also no significant “time” × “group” interaction .The mean amplitudes of the test H-reflex (% of Mmax) for all conditions are summarized in Table n = 10) of the control and conditioned H-reflexes induced by CPN stimulation and TMS stimulation, recorded from one representative subject. Figures F1,14 = 71.43, P < 0.01), but not of “group” . There was significant “time” × “group” interaction . Post hoc analysis indicated that compared to pre, in the visomotor group, the amount of D1 inhibition was significantly increased at post (P < 0.01). A two-way repeated measures of ANOVA for TMS conditioned D1 inhibition showed a significant effect of the “time” , but not of “group” . There was significant “time” × “group” interaction . Post hoc analysis indicated that compared to pre, in the visuomotor group, the amount of TMS conditioned D1 inhibition was significantly increased at post (P < 0.01). In the visuomotor group, the amount of TMS conditioned D1 inhibition was significantly greater than the amount of D1 inhibition at pre (t7 = 4.17 P < 0.01), but not at post . In the non-visuomotor group, the amount of TMS conditioned D1 inhibition was significantly greater than the amount of D1 inhibition at the same period of time . However, there were no significant effects of “time”  and “group”  on the TMS conditioned test H-reflex amplitude, and there was no significant “time” × “group” interaction . Moreover, a two-way repeated measures of ANOVA for the net difference in the amount of D1 inhibition showed a significant effect of “time”  but not of “group” . There was significant “time” × “group” interaction . Post hoc analysis indicated that compared to pre, in the visuomotor task group, the net difference in the amount of D1 inhibition was significantly decreased at post (P < 0.01). The inhibitory effect of D1 inhibition induced by TMS is decreased following the visuomotor task.Figure n = 10) of the control and conditioned H-reflexes induced by TMS stimulation with the C-T intervals at short latency facilitation phase, recorded from one representative subject. The mean amplitudes of TMS conditioned H-reflex at short latency facilitation phase before and after the motor tasks are shown in Figure t7 = 22.21, P < 0.01 in the visuomotor group, t7 = 23.11, P < 0.01 in the non-visuomotor group; post: t7 = 17.77, P < 0.01 in the visuomotor group, t7 = 14.18, P < 0.01 in the non-visuomotor group). There were no significant effects of “time”  and “group”  on the TMS conditioned H-reflex amplitude, and there was also no significant “time” × “group” interaction . The short latency facilitation effects on the H-reflex amplitude did not show any significant difference between pre and post in both groups.Figure t7 = 0.99, P = 0.35 in the visuomotor group; t7 = 0.96, P = 0.37 in the non-visual group), and Hmax/Mmax  following the control task.Following the motor tasks, the mean SOL Mmax was 16.68 ± 1.99 mV in the visuomotor group and 14.44 ± 1.38 mV in the non-visuomotor motor task group, and the mean Hmax/Mmax was 0.45 ± 0.04 in the visuomotor group and 0.49 ± 0.06 in the non-visuomotor group. There were no significant changes in the SOL Mmax (The main findings of our study suggest that: (i) the amount of presynaptic inhibition is increased after visuomotor tasks irrespective of task movement speed; (ii) changes in the reciprocal Ia inhibition are affected by task movement speed, and are increased in fast movement speed conditions, but unchanged in slow movement speed conditions; (iii) non-visuomotor tasks do not induce any changes in presynaptic inhibition and reciprocal Ia inhibition; and (iv) TMS conditioned inhibitory effects on the presynaptic inhibitory pathway are changed following visuomotor tasks.Because muscle fatigue enhances central excitability at the supraspinal levels and changes the presynaptic inhibition  of the H-reflex (Nielsen et al., In the present study, we observed that the presynaptic inhibition was increased following a visuomotor task irrespective of task movement speed, but was unchanged by a non-visuomotor task. Moreover, we also observed the improvement of the task performance among the trials in the slow and fast movement speed conditions. The increase in the presynaptic inhibition of SOL Ia afferent terminals following a visuomotor task was consistent with the results of a previous investigation which demonstrated that presynaptic inhibition was increased after visuomotor tracking tasks involving alternating ankle movement (Perez et al., To determine the influence of descending inputs on the changes in the presynaptic inhibition following a visuomotor task, we examined the TMS conditioning effect of presynaptic inhibition. We showed that the inhibitory effect of presynaptic inhibition induced by TMS was decreased following the visuomotor task, but not following the non-visuomotor task. This noninhibitory effect was not caused by the excitability changes in the corticospinal tract or motoneuron pool; the short latency facilitation effect of TMS stimulation on the SOL H-reflex and Hmax/Mmax were not changed by the visuomotor task or the non-visuomotor task. These results suggest that the changes in presynaptic inhibition observed in the present study are attributed to the activity changes in the interneurons constituting presynaptic inhibitory pathways. It has been shown that the corticospinal tract is essential both for producing spinal plastic changes and for maintaining its changes Wolpaw, . AlthougSeveral studies have reported that H-reflex amplitude is decreased following skilled motor tasks (Perez et al., Training-related changes in the reciprocal Ia inhibition have been studied extensively in human subjects, and these studies have shown that there is also task dependency of the changes in the reciprocal Ia inhibition. For example, the facilitation effect of the reciprocal Ia inhibitory pathway at the onset of ankle dorsiflexion was increased following 4 weeks of explosive isometric dorsiflexion strength training (Geertsen et al., The Ia inhibitory interneurons receive descending inputs from the supraspinal centers that are likely to influence the Ia inhibitory interneuron excitability (Jankowska et al., The changes in the presynaptic inhibition and reciprocal Ia inhibition observed in this study were short lasting after the end of a visuomotor task; the increase in presynaptic inhibition lasted up to 15 min and the increase in reciprocal Ia inhibition lasted up to 5 min after the motor task. These temporary modifications of the spinal neural circuits almost consist with the previous reports (Perez et al., In the present study, all measurements were performed at rest, so it is difficult to see how supraspinal descending inputs are actually responsible for the observed changes in presynaptic inhibition and reciprocal Ia inhibition. Therefore, this should be taken into consideration when interpreting the present results.The results of this study suggest that the supraspinal descending inputs to the spinal cord for controlling joint movement are responsible for the changes in presynaptic inhibition, and that task movement speed is one of the critical factor for inducing activity changes in reciprocal Ia inhibition. These results indicate that spinal neural circuits are differentially modulated, dependent on motor tasks, for achieving the task demands. These task-dependent modulations might be related to the precise control of our limb movements.SK, MH, YK, ST, and KF conceived and designed the research. SK, MH, and YK collected the data. SK analyzed data. All authors participated in the interpretation of the data. SK drafted the manuscript. All authors approved the final version of the manuscript submitted for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.