Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion. Elevated inorganic phosphate levels promote excessive parathyroid hormone secretion, which contributes to the aetiology of secondary hyperparathyroidism. Here, the authors show that phosphate directly inhibits the calcium-sensing receptor, the main regulator of parathyroid hormone secretion. When circulating +2 and Pi from bone2. PTH also stimulates Pi excretion in the renal proximal tubule, thus eliminating the released Pi and so permitting ionized 3. In contrast, increased Pi concentration stimulates PTH secretion by a mechanism that remains unclear7.The homeostatic control of extracellular calcium  respectively, which then increase renal excretion of Pi14.The phenomenology of Pi-induced stimulation of PTH secretion is well described in vitro and in vivo. It has been reported that Pi elicits concentration-dependent stimulation of PTH from bovine2D. SHPT is characterized by parathyroid gland hyperplasia that leads to reduced expression of the vitamin D receptor and CaSR, and chronically elevated PTH secretion. In SHPT, chronic underactivation of the CaSR permits continuously elevated levels of PTH secretion causing chronic dysfunction of the 18. In association with SHPT, increased Ca × P product contributes to vascular calcification and eventual heart disease, calciphylaxis (tissue necrosis), and renal osteodystrophy20. Collectively, these various elements of dysfunctional mineral metabolism are referred to as CKD–MBD , which represents one of the most serious complications of renal disease21. In an attempt to reduce CKD mortality and morbidity, national clinical practice guidelines have been developed24.The molecular mechanism linking Pi and PTH secretion is relevant for understanding the etiology of secondary hyperparathyroidism (SHPT). SHPT is a common complication of chronic kidney disease (CKD), triggered by hyperphosphatemia, hypocalcemia, and low levels of 1,25OH2D supplements, and parathyroidectomy25. However, none of these treatments yet provide sufficient amelioration of CKD–MBD to avoid vascular calcification and cardiovascular mortality26.Currently, the most common therapeutic options for patients with end-stage CKD undergoing dialysis are the calcimimetic drugs cinacalcet or etelcacetide , phosphate binders, 1,25OH427. Of these, sites 1 and 3, based in part on residues R62 and R66, were found exclusively in the inactive conformation, whereas site 4, based partially on residues K225 and R520 was found only in the active conformation. Site 2, based in part on R66 and R69, was observed in both the active and inactive conformations, suggesting a structural role27. These observations suggest that anion binding to sites 1 and 3 may preferentially stabilize the inactive conformation of the CaSR. Here we demonstrate that the CaSR represents a phosphate sensor in the parathyroid gland. Specifically, by increasing extracellular Pi, at concentrations observed in CKD, we demonstrate that hyperphosphatemia inhibits the CaSR in a noncompetitive manner and thus increases PTH secretion. These data provide a molecular mechanism for the stimulatory action of high physiological and pathophysiologic Pi levels on PTH secretion.While the CaSR is the main controller of PTH secretion, its recently crystallized extracellular domain revealed four putative multivalent anion-binding sites occupied by Pi or SO+2 plus either spermine  by measuring CaSR-induced o2+ Fig. . Then, Cine Fig. , NPS-R56ine Fig. . Cells wine Fig. , whereasine Fig. . This inine Fig. . It shoumentclass2pt{minimCarbachol induces non-CaSR-mediated +2, then lowering extracellular pH should reverse this effect by releasing free Ca+2. Therefore, If the apparent inhibitory effect of Pi on CaSR was due solely to buffering ionized Ca27, multivalent anions bind to CaSR at defined sites, distinct from the binding sites described for Ca+2 or l-amino acids. To elucidate the molecular mechanism underlying Pi-mediated CaSR inhibition, we first examined the Emax) to n = 8) and this inhibitory effect was not overcome by further increases in Ca2+ concentration. Interestingly, Pi did not alter 50)   , and also sensitivity to the hyperphosphatemia of CKD, indicating the potential pathophysiological relevance of this effect.According to the recently reported human CaSR-ECD crystal structurells Fig. . As befo50) Fig. . Thus, PmM) Fig.  or with 4 is the other multivalent anion proposed to bind the CaSR ECD according to crystal structure data27. SO4 may act as a ligand at the same sites as Pi, and therefore we tested whether this anion could also inhibit the CaSR. Indeed, SO4 inhibited 50 1.31 mM) , SO4 would appear to be of lesser physiological significance as a CaSR inhibitor. We also tested different Pi concentrations in the presence of 0.3 mM SO4 , and the IC50 obtained was not significantly different from that obtained with Pi alone  Fig. . Howeveri IC50 1.1 mM  in the presence of increasing Pi concentrations  encompassing the physiological and pathophysiological range. A significant increase in PTH secretion was observed at 2 mM Pi (hyperphosphatemia), +56 ± 10%, with a further increase at 3 mM Pi (severe hyperphosphatemia), +77 ± 13%  murine parathyroid glands  of the CaSR. To be considered as full KO Casr, only mice exhibiting (a) high serum PTH levels (greater than tenfold higher than controls), and (b) lack of a response of their parathyroid glands to Ca2+ concentration changes (<10% response when changing from 1.2 to 0.8 mM or from 0.8 to 1.6 mM) were included in the results  content, revealing levels ~17-fold higher than those in control mice, due to the lack of functional CaSR , hereafter termed Pi-binding sites, were identified in the inactive and active CaSR-ECD structures Fig. 27. We exces Fig. . The fouces Fig. . The CaSces Fig. .Fig. 5Pi2+ binding site 3 stabilizes the loop conformation that supports formation of the interdomain bond between R66 and S301. These features highlight the importance of Ca2+ binding site 3 and Pi-binding sites 1 and 3 in regulating CaSR’s activity.To identify the site of Pi-mediated CaSR inhibition, we focused on the two inactive conformation-specific Pi-binding sites 1 and 3 Fig. . In the R62A and CaSRR66A) and the CaSR constructs arising were expressed transiently in wild-type HEK-293 cells. Immunoblotting revealed successful expression of the mutated receptors at levels similar to the WT receptor . The Emax values for these mutants, however, were not altered  to achieve equivalent responses to CaSRWT (0.5 mM Ca2+)  and found that Emax values were not altered in the presence of Pi (P > 0.05)  or efficacy (Emax)  Fig. . We did ax) Fig. .Fig. 7CaR62A was also studied over a more extended range of Pi concentrations (0–5 mM)  increased R62A at any of the Pi concentrations tested (up to 5 mM Pi)  as before  was reduced significantly (by up to 60%), whereas the EC50 for In contrast, we found that CaSRCasr murine parathyroid glands, we reveal that Pi acts as a noncompetitive antagonist of the CaSR at pathophysiological concentrations, resulting in increased PTH secretion. This molecular mechanism may explain the stimulatory effect of Pi on PTH secretion under physiological conditions and in SHPT.Here we identify a mechanism for sensing serum Pi via the CaSR. By combining in vitro signaling experiments with ex vivo PTH secretion measurements from human and KO 2+. Adding 2 mM Pi to the experimental buffer decreased ionized Ca2+ concentration by <7%. However, even when the 2+, Pi still inhibited CaSR activity. In addition, lowering buffer pH to 7.2, equivalent to metabolic acidosis seen in some CKD patients17, failed to overcome the inhibitory effect of Pi, even though it would be expected to reduce the risk of Ca × P formation. These data were consistent with previous observations that pathophysiologic acidosis itself inhibits CaSR31. Together, these findings exclude the possibility that the CaSR-inhibiting effect of Pi seen here can be explained by its potential to lower ionized Ca2+ concentration. Regarding CaSR-induced Emax by 37 ± 3% at 2 mM Pi, without affecting potency (EC50) for 50 of ~1.2 mM, confirming that the CaSR is sensitive to Pi within the physiological range (0.8–1.4 mM). The actions of Pi at concentrations >1.5 mM are relevant for CKD whose cardinal features include hyperphosphatemia (>2 mM). Indeed, when CaSR was stimulated with cinacalcet, a drug widely used in dialysis patients, 2 mM Pi reduced the CaSR response by 46 ± 8%. Therefore, variations in serum Pi concentration might explain some of the variability of cinacalcet effectiveness observed across CKD patients25, though it should be noted that these values were obtained by using CaSR-HEK cells, whereas CaSR sensitivity for Ca2+, R568, and Pi differs in parathyroid cells.We first characterized the effects of Pi on CaSR-mediated signaling, demonstrating that raising Pi concentration from 0.8  to 2 mM (CKD-like) significantly inhibited maximal levels of 27, SO4 also attenuated CaSR activity in the same range as Pi; however, as physiological SO4 concentrations are lower (~0.3 mM in serum), SO4 is less likely to be a physiologically relevant CaSR inhibitor. On the other hand, plasma SO4 levels can also be elevated in CKD and therefore might also contribute to SHPT32.Consistent with CaSR-ECD structural data34. We tested our hypothesis by measuring PTH secretion from freshly isolated human parathyroid cells ex vivo in a perifusion model. PTH secretion experiments were usually conducted within 24 h (maximum 48 h) from surgical excision and tissue was stored undigested at 4 °C to minimize the loss of CaSR expression reported in previous studies35. Parathyroid cell preparations were initially exposed to low Ca2+ concentrations to induce PTH secretion and to confirm CaSR expression and its functional responsiveness. Then, the parathyroid cells were exposed to small increases in Pi concentration, within the CKD range, and a rapid (within 2 min) and quickly reversible rise in PTH secretion consistent with a cell surface receptor-mediated action was observed that cannot be explained by genomic changes alone. These results are consistent with a previous in vivo study performed in rats in which duodenal or intravenous Pi injections elicited rapid (within 10 min) increases in plasma PTH, and the authors hypothesized the existence of a cell surface “phosphate sensor”, similar to the Ca2+ sensor, and expressed in the duodenum and the parathyroid gland36. An alternative hypothesis for the progression of SHPT is that partial downregulation of parathyroid CaSR expression occurs, permitting increased PTH secretion and this would mean fewer CaSRs for Pi to inhibit. However, one of the leading treatments for SHPT in advanced renal disease are the calcimimetics , which target the CaSR directly and provide clinically significant reductions in PTH secretion. Therefore, even in advanced CKD, there remains sufficient CaSR expression to regulate PTH secretion37. Furthermore, Pi also rapidly stimulated PTH secretion in control mice parathyroid glands, even though physiological serum Pi levels in mice are higher than those in humans (+0.6 mM)38. In contrast, in parathyroid-specific KO Casr mice, which lacked Casr mice exhibit elevated PTH secretion and therefore the potential for further substantial increases in secretion may be impaired.The association of increased Pi concentration with both excessive PTH secretion and parathyroid gland hyperplasia has been widely demonstrated in vitro and in vivo although the causal link remains uncertain27. However, a different CaSR-ECD crystal structure obtained by Zhang et al. did not reveal any potential Pi-binding sites, although Pi was not present in the crystallization buffer39. From Geng’s crystal structure model, we assessed which putative anion-binding site(s) were most likely to mediate functional inhibition, based on their presence in the inactive and active conformations. Anion-binding site 1, based in part on R62, and site 3, based in part on R66, were present solely in the inactive conformation. Thus, we focused on R62 and R66, which because of their positively charged side chains interact directly with Pi ions. The loss of function displayed by CaSRR62A and CaSRR66A can be explained by impairment of R62–E277 and R66–S301 interactions occurring between the upper and lower domain, as they both support receptor stability in the closed (active) conformation. The crucial finding, however, was that R62 appears to be the principal residue accounting for anion-dependent inhibition. Specifically, CaSRR62A exhibited no anion-induced inhibition of CaSR in either R62A-HEK cells. The R62–E277 salt bridge that connects upper and lower domains, holds the receptor in its active conformation 27, which is impaired in this mutant and enhances domain closure for receptor activation. However, increased CaSR activity with low Pi was not seen in CaSRWT, as Ca2+ binding site 3 and R62–E277 already stabilize this loop conformation. In contrast, CaSRR66A was inhibited by Pi in the same way as CaSRWT, pointing to a primary inhibitory role of R62 upon Pi binding, and a primary role of R62–E277, rather than R66–S301, in stabilizing the active conformation of the receptor. Further, the CaSR Pi-binding sites exhibit a high degree of amino acid sequence conservation across species, largely absent from other family C GPCRs, suggesting an important role for these sites in CaSR biology.The idea that CaSR is a Pi sensor is supported by the crystal structures of the human CaSR ECD, obtained by Geng et al., which revealed four multivalent anion-binding sites2+ homeostasis and for CKD. We propose that Pi regulates the active–inactive equilibrium of CaSR such that at higher plasma Pi concentrations, the CaSR equilibrium is displaced toward its inactive conformation, thus permitting increased PTH secretion 22. Therefore, it will be interesting to determine whether strategies aimed at reducing extracellular Pi concentrations in CKD patients with SHPT might enhance the effectiveness of calcimimetics  in lowering serum PTH levels.In the context of CKD, where serum Pi levels are increased due to the impaired renal excretion, the homeostatic response described above could produce continuously elevated PTH secretion leading to SHPT, bone loss, and further hyperphosphatemia. In addition to hyperphosphatemia, there are other factors that also contribute to the development and progression of SHPT in the context of CKD: low 1,25OH47. Therefore, such hyperphosphatemia might moderate the gain of function by partially inhibiting the CaSR, so limiting the severity of the hypocalcemia, hypercalciuria, and nephrocalcinosis. Indeed with CaSR expressed in numerous tissues throughout the body, our findings may provide a more general Pi-sensing mechanism for the maintenance of extracellular Pi, for example in CaSR-expressing osteoblasts or gut cells where Pi has direct effects.There is also a possibility that Pi may elicit a protective role in patients with autosomal-dominant hypocalcemia. These patients express activating mutations in CaSR, or in G protein 11 (GNA11), which cause hypocalcemia, inappropriately low PTH levels, and moderate hyperphosphatemiaIn summary, these results provide an original molecular mechanism that explains Pi-stimulated PTH secretion and further provide a mechanism for sensing elevated Pi concentrations in CaSR-expressing tissues. Then in the hyperphosphatemia of CKD, Pi-mediated stabilization of the CaSR’s inactive conformation would elicit chronically increased PTH release as the damaged kidneys fail to excrete the Pi, leading to SHPT, and in turn to further bone loss and hyperphosphatemia Fig. .2 and 0.9 mM Na2HPO4 from Sigma (D5796), and fortified with 10% fetal bovine serum (FBS). Cells were dissociated with trypsin upon reaching 80–90% confluence and grown at 37 °C with 5% CO2 until passage 30.HEK-293 cells (from ATCC) were used as a model for the functional expression of CaSR protein. Wild-type HEK-293 and stably transfected CaSR-HEK-293 (CaSR-HEK) cells were grown in Dulbeccos’ modified Eagle’s medium containing 1.8 mM CaCl2, 0.5 MgCl2, and 5.5 glucose with Pi added as Na2HPO4 and KH2PO4 in a 4:1 ratio (pH 7.4). Following equilibration (20 min), cells were treated for 10 min with experimental buffer including different Pi concentrations and then lysed on ice for 15 min in a RIPA-like buffer supplemented with protease and phosphatase inhibitors ((mM) 12 HEPES (pH 7.4), 300 mannitol, 1 EGTA, 1 EDTA, 1% (v/v) Triton-X 100, 0.1% (w/v) sodium dodecyl sulfate (SDS), 1 NaF, 250 μM sodium pyrophosphate, 100 μM sodium vanadate, 1.25 μM pepstatin, 4 μM leupeptin, 4.8 μM PMSF, and 1 N-ethylmaleimide). Phosphorylation of ERK (pERK) was quantified by semiquantitiative immunoblotting.CaSR-HEK cells were grown on 30-mm dishes and then incubated at 37 °C in experimental buffer containing (mM) 20 HEPES (pH 7.4), 125 NaCl, 4 KCl, 0.5 CaClβ Actin-Peroxidase monoclonal (AC-15) mouse antibody  from MERCK (Cat. number A3854)] for 1 h. Secondary antibody was horseradish peroxidase-conjugated horse anti-mouse IgG  from Cell Signaling (Cat. number 7076). Chemiluminescence was generated by using BIO-RAD clarity western ECL substrate, detected by using a Chemidoc gel analyzer (BIO-RAD), and quantified by densitometry by using Image Lab 6. Uncropped and unprocessed immunoblot scans shown in Figs. Protein samples were first diluted (4:1) with 5× Laemmli buffer followed by resolution of the proteins by SDS-PAGE and electrophoretic transfer into a nitrocellulose membrane . Membranes were incubated in blocking buffer solution containing Tween-TBS solution and 5% (w/v) bovine serum albumin (BSA) for 1 h and probed with primary antibodies for pERK [Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse (1:3000 dilution) from Cell Signaling (Cat. number 9106)], CaSR [Calcium Sensing Receptor monoclonal mouse antibody  (1:3000) from Thermo Fisher Scientific (Cat. number MA1-934)], or β-actin  and placed in the middle of a 13-mm track-etched (0.1 μM pore) polycarbonate membrane (Whatman), floating on a large drop (500 μl) of ice-cold secretion media supplemented with 3 mM Ca2+ 51. The time used to find, dissect, and clean the parathyroid glands from each mouse was <5 min per animal. The dissected glands were equilibrated in fresh secretion media (at 37 °C) for 1 h. Then, each pair of glands were incubated for 30 min in 500 μl of secretion media (37 °C) containing different concentrations of Ca2+ and Pi. Intact PTH (1–84) secreted into the bathing culture media was then collected and quantified by ELISA assay (Immunotopics Inc.) and expressed as pg PTH/30 min/gland. Mouse serum was also collected and assayed for intact PTH. Only KO Casr mice displaying high serum PTH levels (more than tenfold higher than control) and whose excised parathyroid glands were insensitive to Mice with homozygous parathyroid gland-specific deletion of exon 7  of the CaSR  was used to visualize amino acid conservation in the four Pi-binding sites predicted by Geng et al.27. Sequence conservation by position was assessed by bit-score calculation for each amino acid residue position in the four Pi-binding sites, with the relative frequency of each amino acid displayed pictorially by height of letter.A data set of family C GPCR alignments was generated by Herberger et al.55.CaSR-ECD original structures were obtained from Protein Data Bank (PDB) by using the accession numbers 5K5S (active conformation) and 5K5T (inactive conformation). Anion-binding interactions were studied by using PyMol Molecular Graphics System, Schrodinger56. Responses were measured for 190 s, but excluding the first 40–50 s to avoid change artifacts. Densitometry of immunoreactive bands was performed by using Image Lab 6.0 2D software (BIO-RAD). GraphPad Prism 7  was used for nonlinear regression analysis, mean, and SEM. Orthosteric concentration−response curves were normalized to 100% as the maximum orthosteric agonist/antagonist response. Normalized data were fitted to a four-parameter concentration response for Changes in Fura2 ratio (340/380) were measured with MetaFluor imaging system and assessed by determining the area under the curve by using the R-studio “PKNCA Perform Pharmacokinetic Non-Compartmental Analysis” package50 and Emax in the presence and absence of Pi were determined by an extra sum-of- squares F-test. A one-way ANOVA with Dunnett’s multiple comparisons post test was used to determine statistical differences between EC50 and Emax. A one-way repeated measure ANOVA, with Dunnett’s for normally distributed data or Friedman Dunn’s for non-normally distributed data, was used to determine statistical differences between 0, 0.8, and 2 mM Pi in t test. Data are presented as mean ± SEM or median with range, except for EC50 and IC50 values, which are presented as mean 95% confidence interval, and P values <0.05 were considered statistically significant. Replicates are reported either as n for the number of coverslips used per experiment, or as N for the number of independent experiments or animals.Statistical differences between the ECFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting SummarySource Data


The results showed that the type of inhibition with glyceollin was competitive, that of genistein was noncompetitive, that of daidzein was uncompetitive, and luteolin showed a mixed mode of action. The iK values were determined using a Dixon plot as glyceollin, 18.99 μM; genistein, 15.42 μM; luteolin, 16.81 μM; and daidzein, 9.99 μM. Furthermore, potential synergistic effects between glyceollin and the three polyphenols were investigated. A combination of glyceollin and luteolin at a ratio of 3:7 exhibited synergistic effects on α-glucosidase inhibition, having a combination index (CI) of 0.64244, according to the CI–isobologram equation. Collectively, these results showed that a combination of glyceollin and luteolin has the potential to inhibit α-glucosidase activity via a synergistic mode of inhibition.To determine the mechanism of action of the effects of phytoalexins in soybeans, we analyzed  Soybeans are an important food resource that has beneficial purposes as “meat in the field” because of its nutritional value . PresentAspergillus sojae. However, data on the enzymatic effects of glyceollins derived from phytoalexins and studies on how glyceollins and other anti-diabetic agents are involved in the inhibition of α-glucosidase are lacking.Glyceollins, which are synthesized from daidzein in soybeans infected with fungi, are potent antioxidants that inhibit the production of reactive oxygen species (ROS) . Glyceolp-nitrophenyl α-D-glucopyranoside (pNPG), and there is mounting evidence that various α-glucosidase inhibitors produced by plants such as legumes also exert anti-diabetic effects  are the maximum reaction velocity and the substrate concentration, respectively; and mK and sK are the Michaelis–Menten and dissociation constants for the affinity of the substrate, respectively. The α symbol is the ratio of the uncompetitive inhibition constant to the competitive inhibition constant and has a value of 1 for noncompetitive inhibition. The iαK value is the inhibitor constant when the inhibitor (I) occupies the enzyme–substrate (ES) complex p < 0.05.The results are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. Statistical differences were determined by Tukey’s one-way ANOVA using IBM SPSS software . A difference was considered significant at 50) of acarbose, glyceollin, genistein, luteolin, and daidzein were 530.50 ± 100.13 μM, 13.22 ± 2.31 μM, 23.66 ± 3.54 μM, 11.94 ± 1.63 μM, and 20.16 ± 6.17 μM, respectively. These results show that the four soybean-derived polyphenol compounds exert α-glucosidase inhibitory activity that is more than 20 times higher than that of acarbose.The ability of polyphenol compounds derived from soybeans to inhibit α-glucosidase B–E was mmax) for α-glucosidase was calculated from the nonlinear regression curve with five different concentrations of substrate  versus the substrate concentration  was plotted. A single regression line for each concentration of the substrate was obtained, and iK was calculated from the intersection of the four lines. The iK values were determined by GraphPad Prism 6.0 software as 45.88 ± 3.75 μM, 18.99 ± 4.45 μM, 15.42 ± 2.48 μM, and 16.81 ± 9.60 μM, for acarbose, glyceollin, genistein, and luteolin, respectively  is −10.3 kcal/mol. This structural model illustrates the competitive inhibition of glyceollin. Although the polyphenols tested are similar in structure, they dock in the active site pocket with different orientations. Interestingly, the model with daidzein shows that the substrate covers the entrance of the pocket with a ΔGbind of −7.6 kcal/mol –isobologram equation that allows quantitative determination of compound interactions, where CI < 1, CI = 1, and CI > 1 denote synergism, an additive effect, and antagonism , respectiK values; however, we do not know how the structure of the enzyme is changed by the binding of inhibitors. Therefore, it would be useful to perform molecular docking simulation studies and protein structure analyses, including X-ray crystallography and nuclear magnetic resonance (NMR), to determine the mode of synergistic inhibition of soybean-derived polyphenols, which will shed light on their inhibitory mechanisms.To verify whether the combination of glyceollin and luteolin affects the mode of α-glucosidase inhibition, it was necessary to analyze the affinity of each of the polyphenol compounds. When soybeans are infected with various elicitors, polyphenol compounds are naturally produced. Such polyphenol compounds are called phytoalexins, although the identification of the polyphenols is still needed. This study investigated the synergistic potency of two polyphenols by comparing their α-glucosidase inhibition, showing that a combination of glyceollin and luteolin has the potential to inhibit α-glucosidase activity via a synergistic mode of action. The inhibition of α-glucosidase by polyphenol compounds from soybeans is not so high when the soybeans are not elicited by Aspergillus. However, the glyceollin produced by fermentation in response to Aspergillus or other fungi is thought to rapidly enhance the antidiabetic effect. Collectively, we believe that fermentation of soybeans induces various phytoalexins ; therefore, the intake of fermented soybeans in foods such as soy sauce, soy paste, Koji, and Natto might be useful to synergistically prevent and/or control type 2 diabetes mellitus.By analyzing enzyme inhibition kinetics using Michaelis–Menten plots and Lineweaver–Burk plots, we found that inhibition mode of glyceollin was competitive inhibition, that of genistein was noncompetitive, that of daidzein was uncompetitive, and luteolin showed a mixed mode of action. These results indicate that glyceollin, genistein, luteolin, and daidzein could be promising α-glucosidase inhibitors for anti-diabetic approaches. A combination of glyceollin and luteolin had synergistic effects on


To the Editor,neu indicated that IgE could complement or possibly improve the clinical performance of trastuzumab.http://www.clinicaltrials.gov).Monoclonal antibodies approved for the treatment of cancer belong to the IgG class (most often IgG1). However, IgG has limited tissue half‐life 2‐3 days), relatively low affinity for cognate Fc receptors and the disadvantage of interaction with inhibitory Fcγ receptors, abundant in the tumour microenvironment. Conversely, IgE class antibodies may offer new options for cancer therapy, based on high affinity for cognate Fcε receptors expressed on different, often tumour‐resident, immune effector cells such as macrophages and mast cells, and lack of inhibitory Fc receptors.‐3 days, Despite considerable progress, production of monoclonal antibodies remains time‐consuming and labour‐intensive. One reason is the requirement for expression of heavy (HC) and light chains (LC) in a controlled manner, usually cloned in separate expression vectors using enzymatic restriction digestion and ligation. This introduces experimental variability in expression procedures and is often inefficient. These limitations also concern the study of anti‐allergen IgE, where Fabs rather than full‐length antibodies are commonly expressed and evaluated.Building upon ours and others' previous methodologies, we report the efficient transient expression and functional evaluation of IgE, exemplified using the variable region sequences of trastuzumab and human IgE constant regions (anti‐HER2 IgE).We employed polymerase incomplete primer extension (PIPE) PCR cloning and enzyme‐free assembly of DNA fragments. The amino acid sequences of trastuzumab variable light (VL) and heavy (VH) chain regions were manually codon‐optimized for a human expression host and cloned into a pVitro1‐hygro‐mcs dual‐expression vector containing precloned cassettes of the human epsilon HC and kappa LC using PIPE PCR cloning methodology  cells without antibiotic selection, in 30 mL serum‐free suspension cultures Figure C. Variabneu‐overexpressing  breast cancer cells and moderately expressing MCF‐10 normal breast cells, and its HER2 antigen recognition kinetic profile on tumour cells was comparable to trastuzumab  or by HER2‐expressing tumour cells (right), but not without cross‐linking stimulus or with recombinant monomeric antigen , readily established even in “small” environments, surpassing previous platforms in expression efficiency, speed (7‐9 days vs 4‐6 weeks) and yields (70‐80 mg/mL vs <20‐25 mg/mL),SN Karagiannis and JF Spicer are founders and shareholders of IGEM Therapeutics Ltd. SN Karagiannis holds a patent on antitumour IgE antibodies. All other authors have declared that no conflict of interest exists.The study is a result of a collaborative effort from the AllergoOncology Task Force of the European Academy of Allergy and Clinical Immunology (EAACI). The authors acknowledge support by Breast Cancer Now (147), working in partnership with Walk the Walk; Cancer Research UK ; the Medical Research Council (MR/L023091/1); the Academy of Medical Sciences; CR UK//NIHR in England/DoH for Scotland, Wales and Northern Ireland Experimental Cancer Medicine Centre (C10355/A15587). The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) based at Guy's and St Thomas' NHS Foundation Trust and King's College London (IS‐BRC‐1215‐20006). Further funding: Austrian Science Fund in the frame of the Doctoral Program BioToP (Grant W 1224) . JF‐S and EJ‐J were supported by the Austrian Science Fund, projects W1205‐B09  and in part SFB F4606‐B28. JF‐S is a recipient of an EAACI  Exchange Research Fellowship 2016 and a Boehringer Ingelheim Fonds Travel Grant 2017. The authors are solely responsible for study design, data collection, analysis, decision to publish and preparation of the manuscript. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Click here for additional data file.


We developed Supportive care Prioritization, Assessment and Recommendations for Kids (SPARK), a web-based application designed to facilitate symptom screening by children receiving cancer treatments and access to supportive care clinical practice guidelines primarily by healthcare providers. The objective was to describe the initial development and evaluation of SPARK from the perspective of children.Development and evaluation occurred in three phases: (1) low fidelity focused on functionality, (2) design focused on “look and feel” and (3) high fidelity confirmed functionality and design. Cognitive interviews were conducted with children receiving cancer treatments 8–18 years of age. Evaluation occurred after every five interviews and changes were guided by a Review Panel. Quantitative evaluation included SPARK ease of use and understandability of SPARK reports.n = 30), design (n = 30) and high fidelity (n = 30). Across phases, the median age was 13.2 (range 8.5 to 18.4) years. During low-fidelity and design phases, iterative refinements to SPARK improved website navigation, usability and likability from the perspective of children and established symptom report design. Among the last 10 children enrolled to high-fidelity testing, all (100%) understood how to complete symptom screening, access reports and interpret reports. Among these 10 respondents, all (100%) found SPARK easy to use and 9 (90%) found SPARK reports were easy to understand.The number of children included by phase were: low fidelity (SPARK is a web-based application which is usable and understandable, and it is now appropriate to use for research. Future efforts will focus on clinical implementation of SPARK. Children receiving cancer treatments have excellent survival outcomes, in part, related to the provision of intensive therapies. Unfortunately, most children suffer and experience severe and bothersome treatment-related symptoms . Common In order to facilitate systematic symptom screening, a tool appropriate for children with cancer is required . We thusIn addition to the development and evaluation of SSPedi, we also appreciated the need to facilitate symptom management through improving access to clinical practice guidelines for supportive care needs. Clinical practice guidelines are recommendations based on systematic reviews of the evidence and an assessment of the benefits and harms of different strategies that aim to improve patient care . GuideliIn order to bring symptom screening and access to clinical practice guidelines together toward improving the supportive care of children with cancer and pediatric hematopoietic stem cell transplantation recipients, we developed Supportive care Prioritization, Assessment and Recommendations for Kids (SPARK). SPARK is a web-based application that consists of two components: (1) a symptom screening component centered on SSPedi, and (2) a supportive care clinical practice guideline component. SPARK tracks symptoms over time, allows patients, family members and health care professionals to view symptom reports, and facilitates access to supportive care clinical practice guidelines. For SPARK to have clinical utility, it has to be useable and liked from the perspective of the intended users. The objective was to describe the initial development and evaluation of the symptom screening aspect of SPARK from the perspective of children receiving cancer treatments.SPARK pages were initially developed by the research team and a web application development company, Translucent Computing Inc. While SPARK is designed for use by pediatric patients, family members and healthcare providers, this study focused on pages aimed at pediatric patients . These pages were: 1) the landing page Fig. , (2) acc the landThe landing page Fig.  includesDevelopment and evaluation of SPARK occurred in 3 phases: (1) low fidelity, (2) design and (3) high fidelity. The main purpose of the low-fidelity phase was to evaluate and enhance the functionality of SPARK web pages, concentrating on how to access and complete SSPedi, navigate between different pages and view reports. All pages were in black and white . The main purpose of the design phase was to attain the desired “look and feel” of SPARK. All pages were in color. The design phase was divided into two sub-phases, namely SSPedi report design and overall design. The main purpose of the high-fidelity phase was to evaluate and confirm both functionality and design. This phase was divided into two sub-phases, namely coding sprints and finalization. The phases and sub-phases are detailed under Procedures below.We used the Technology Acceptance Model as the guiding principle behind this research. This model states that perceived ease of use and perceived usefulness are predictors of behavioral intention and actual use of health technology . ConsequWe included English-speaking children and adolescents with cancer and pediatric hematopoietic stem cell transplant recipients who were 8–18 years of age at the time of enrollment. Exclusion criteria were illness severity, cognitive disability or visual impairment that precluded utilization of SPARK according to the primary healthcare team. Children were purposively sampled to maximize variation by age group, gender and underlying diagnosis. All participants were recruited from The Hospital for Sick Children in Toronto, Canada.For each phase, both quantitative and qualitative data were collected and used to make decisions about development. Draft versions of SPARK were reviewed and approved by a Review Panel composed of two pediatric oncology survivors (ND and HD), one parent advocate (SK), a pediatric psychologist (BS), a behavioral scientist (DS), a pediatric pharmacist (LD) and a pediatric oncologist (LS). Behavioral science was used as an input into choice architecture, which is the process of designing information and choice context to nudge individuals toward making better decisions .In general, participants were first given the opportunity to explore the website freely and then asked to complete specific tasks. The think aloud method was incorporated within the interviews to evaluate different aspects of SPARK . ParticiThe purpose of this phase was to evaluate and enhance the functionality or usability of SPARK web pages. All pages were in black and white  to determine whether participants understood how to use SPARK without distraction by color, since addition of color would introduce preferences for a specific design.First, we had to identify an appropriate patient icon and terminology to refer to patients. Thus, for the first 20 participants, we identified preference for the patient icon among four choices Fig. ; we randhttps://www.invisionapp.com/). Clickable areas on each image mimicked that of a functional website. We gave participants time to navigate through the different patient-specific web pages of SPARK. After free-exploring, we asked participants how they would perform specific tasks such as completing SSPedi (Completing SSPedi) or seeing their current or previous SSPedi scores (Seeing current or previous SSPedi score). Next, we evaluated understanding of each patient-specific page and asked what each page, element and icon meant. From the landing page, we evaluated understanding of the overall page  and each of the child, family member and healthcare provider icons . If the user scrolls to the bottom of the landing page, there is an explanation of SPARK and SSPedi; this section was also evaluated (What are SPARK and SSPedi). We then evaluated understanding of the “Do SSPedi Now!” page (Do SSPedi Now!) and the “How Will SSPedi Help Me?” page (How Will SSPedi Help Me?).To make it clear what sections were specifically evaluated for understanding, the “label” used in the Results follows the task description in bold and parenthesis. Low-fidelity evaluation was conducted using a mock version of the website that was created by uploading an image of each page to InVision ; and (3) specific symptom longitudinally with worsening over time (Interpreting specific symptom worsening over time). Participants were asked to interpret the vertical and horizontal axes, find specific scores on a specific date  and to interpret each report.From the “My SSPedi Scores” page, we asked participants to evaluate three hypothetical reports: (1) single SSPedi administration showing degree of bother of 15 symptoms  focused on how best to display a single SSPedi administration report . The additional pages were the landing page, access to SSPedi, information about how SSPedi helps, and trends over time graphs showing a specific symptom (throwing up). We assessed understanding and preferences of pages and elements such as icons, pictures and colors.The final phase was high fidelity, in which both functionality and design were evaluated and confirmed. Procedures mirrored those of low fidelity except that the live SPARK website was evaluated instead of the mock InVision version. This phase was divided into two sub-phases, namely coding sprints and finalization. Coding sprints (cohorts 8 and 9) focused on a sub-set of SPARK pages and ensured that respondents could access SSPedi, complete SSPedi, retrieve instructions, cancel out of SSPedi and save SSPedi scores. The last sub-phase was finalization (cohort 10) and it evaluated the entire patient-facing portal of SPARK. As with low-fidelity evaluation, we asked questions about the website overall including ease of use of SPARK, ease of understandability of reports, and usefulness for future children receiving cancer treatments.Throughout each phase, we evaluated responses after every five interviews and a smaller research team  met to decide whether the script or SPARK required minor edits. After every 10 to 20 interviews and when the smaller research team was satisfied that a phase had been completed , the Review Panel met and could request further edits  or confirm completion of a phase.Quantitative evaluation in low- and high-fidelity phases included understandability, usability, ease of understanding of SPARK reports and usefulness for future children receiving cancer treatments. Understandability was rated externally by experts (two interviewers) while usability, ease of understanding of SPARK reports and usefulness were rated by the user-patient (children themselves). More specifically, understandability of SPARK elements were evaluated by the two interviewers on a 4-point Likert scale ranging from 1 = “completely incorrect” to 4 = “completely correct”. The two interviewers rated understandability independently and then compared ratings. If they disagreed, they referred to field notes to arrive at consensus. Usability of the SPARK website was evaluated by the participant on a 5-point Likert scale ranging from 1 = “very hard” to 5 = “very easy”. Ease of understanding of SPARK reports was similarly rated. Usefulness for future children receiving cancer treatments was evaluated by the participant on a 5-point Likert scale ranging from 1 = “not useful at all” to 5 = “very useful”. We reported the proportion of participants who were correct or completely correct, found SPARK/SPARK reports easy or very easy to use or understand and thought SPARK would be useful or very useful for future children receiving cancer treatments.Criteria for completion of low-fidelity and high-fidelity  phases were based upon quantitative and qualitative responses as follows: (1) saturation (absence of new themes); (2) at least 9 of the last 10 participants stated the website was easy or very easy to use; and (3) qualitative comments did not suggest that further modifications were required. Based upon previous experience, we anticipated conducting between 6 and 12 iterations with 5 participants per iteration for a total of 30 to 60 participants for each phase, or 90 to 180 total interviews.Figure Table There were 15 respondents included in the SSPedi report design sub-phase in which the single SSPedi administration report  was evaluated, and 15 respondents in the overall design sub-phase in which the five developed pages were evaluated. For the first 10 patients in the SSPedi report design sub-phase (cohort 4), 7 (70%) preferred the single SSPedi administration report to be laid out with horizontal symptom score bars such that different symptoms were presented along the Y-axis  understood how to access and complete SSPedi, 19 (95%) were able to retrieve SSPedi instructions, 20 (100%) understood how to cancel out of SSPedi and 20 (100%) understood how to save scores. Based upon qualitative comments, minor edits were made to the SPARK pages leading to the finalization sub-phase. Understanding (rated as mostly correct or completely correct) was perfect (100%) for all tasks, pages, icons and reports in cohort 10 .Project home page:https://www.sungresearch.com/spark/Operating system: Platform independent.Other requirements: NA.Programming language: Java.License: NA.Any restrictions to use by non-academics: Currently only available for use in research through The Hospital for Sick Children.


The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation.Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively.Purified echicetin from snake IIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates  and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin−/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways.EB-induced platelet aggregation was dependent on integrin αEB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα−/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.The online version of this article (10.1186/s12964-019-0428-1) contains supplementary material, which is available to authorized users. As the smallest circulating blood cells, platelets are activated at sites of vascular injury resulting in adhesion, aggregation, and blood coagulation, thereby preventing major blood loss. Pathologically, platelet hypofunction and hyperfunction can result in life-threatening bleeding or thromboinflammatory disorders, respectively. Platelet activation responses with subsequent thrombus formation are tightly controlled by multiple factors, which promote or inhibit platelet activation via membrane receptors and their intracellular effector systems. An essential platelet receptor is the glycoprotein (GP) Ib-IX-V complex, and its subunit GPIbα ligates predominantly von Willebrand factor but also other adhesion proteins and distinct coagulation factors. This enables platelet recruitment to the vessel wall, aggregation and coagulation. Despite this important role, intracellular effects of GPIb-IX-V in platelets and their interaction with other signaling pathways are not well defined. Recently, we characterized the snake venom protein echicetin immobilized on polystyrene beads as a specific GPIb activator. With this tool, we now show that GPIbα causes the activation of the tyrosine kinase Syk in a Src family kinase dependent manner, which results in the generation of further intracellular messengers and ultimately platelet aggregation. Surprisingly, activation of the platelet inhibitory cAMP/PKA and cGMP/PKG pathways enhanced initial Syk phosphorylation/activation, but strongly inhibited GPIbα-induced platelet aggregation, which is distal to Syk activation. These studies establish a new intracellular regulatory network triggered by the activation of GPIbα in human platelets.Circulating platelets are essential for both physiological and pathological hemostasis and have important roles in inflammatory diseases and cancer –3. Plate2 (TxA2)] and adhesion molecules  bind to and stimulate specific G-protein-coupled receptors (GPCRs), or cell membrane-spanning adhesion receptors, respectively. These receptors stimulate intracellular signaling pathways and various platelet responses leading to integrin activation (e.g. integrin αIIbβ3), granule secretion, exposure of anionic phospholipids -2-oxo-1H-indole-5-sulfonamide were purchased from Merck, Germany and PRT060318 (PRT-318), -2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide), was from Selleckchem, Germany. PP2 and its inactive analogue PP3 were from Abcam, England. Tirofiban (Aggrastat®) was from Iroko Cardio LLC, USA. MRS2179 was from Viozol, Eching, Germany, AR-C69931 from The Medicines Company, Parsippany, NJ, USA and SQ-29548, was from Cayman chemical, MI, USA. Wortmannin was purchased from Biozol, Eching, Germany. Iloprost (Ilomedine®) and riociguat were from Bayer, Germany. Bovine serum albumin (BSA) was from Capricorn Scientific GmbH, Germany. Clarity™ Western ECL Substrate was from BioRad Laboratories, USA. Rabbit monoclonal antibodies against phosphorylated Syk Y525/526 and polyclonal antibodies against phosphorylated Syk Y352, LAT Y191, PLCγ2 Y759, Akt S473, VASP S239, α-actinin were from Cell Signaling Technologies, USA. Mouse monoclonal antibodies against Syk, PLCɣ2 and Akt were from Santa Cruz Biotechnology, USA. Antibodies against β-actin were from Abcam, Engalnd. Anti- phosphotyrosine mouse antibodies were from Merck, Schwalbach, Germany. Secondary antibodies HRP-conjugated goat anti-rabbit and anti-mouse IgG were from BioRad Laboratories Hercules, USA. IP-One ELISA kit (96 wells) was from Cisbio, Codolet, France. Fluo-3, AM, intracellular cytoplasmic Ca2+ indicator was from Life Technologies, USA.Lyophilized snake venom of E. carinatus sochureki lyophilized venom by affinity chromatography followed by DEAE anion exchange chromatography and validated by mass spectrometry analysis. For affinity chromatography protein A sepharose-4B column coated with rabbit polyclonal antibodies directed against echicetin (generated by A. Navdaev) was used. Echicetin was eluted using 0.2 M acetate buffer pH 2.7. The eluent buffer was exchanged into 10 mM Tris buffer pH 8.0 (buffer A) and then applied to DEAE anion exchange column. Elution of echicetin was performed by a 0 to 1 M gradient of NaCl in buffer A, under a flow rate of 1 ml/min. Fraction eluted at 120 mM NaCl consisted equally of α and β subunit and was used in all the experiments. Silver staining and mass spectrometry analysis were performed in order to confirm the purity of echicetin. Echicetin beads (EB) were prepared as reported [Echicetin was purified from reported  and coatSamples from peak 1 and peak 2 were prepared under reducing conditions (by adding Laemmli buffer) then boiled at 95 °C for 10 min. Proteins of both peaks were separated by electrophoresis using 15% SDS-PAGE gels. Gels were stained using InstantBlue™. Bands were cut and digested using trypsin. Protein sequences were analyzed by mass spectrometry in the mass spectrometry core facility at the University Medical Center of the Johannes Gutenberg University, Mainz.ITGA2B c.621C > T; p.T176I [2, 10 mM Glucose, 10 mM Hepes) pH 7.4. Agglutination followed by fibrinogen-dependent aggregation of washed human platelets was induced by adding human vWF  plus ristocetin  as previously described [8 platelets/ml) were pre-incubated at 37 °C with different inhibitors: Syk inhibitors, OXSI-2 or PRT-318, or Src family kinase inhibitor PP2 or its inactive analogue PP3 or wortmannin. In addition, ADP and TxA2 receptors blockers were used , cAMP-elevating agents iloprost and cGMP-elevating soluble guanylate cyclase (sGC) stimulator riociguat, respectively. Platelet aggregation was triggered with 0.5% (v/v) echicetin beads for 1, 2 and 5 min under continuous stirring (1000 rpm) at 37 °C in an Apact4S Plus aggregometer . Control samples mentioned as basal (in graphs) or time zero (in blots and graphs) refer to washed platelets placed under continuous stirring for 1 min in absence of any agonist.Venous blood was collected as citrated whole blood after informed consent from healthy volunteers who did not take any medication for at least 10 days before blood collection. Studies using human platelets from healthy volunteers and from a patient with Glanzmann thrombosthenia caused by a homozygous point mutation in  p.T176I , 43 wereescribed . Residua(PVDF) membranes before blocking the membranes for one hour with 5% BSA in TBS-T  at room temperature. Membranes were incubated overnight under gentle shaking with 1:1000 diluted primary antibodies. Membranes were washed three times with TBS-T and incubated for two hours at RT with the appropriate secondary antibodies diluted 1:5000 in 5% BSA. Membranes were washed again for 3 times with TBS-T before developing the blots by ECL detection. Control blots with total proteins  derive from same samples from the corresponding phosphorylated form. α-actinin was also used as loading control for distinct p-Akt blots. Blots with p-LAT, p-VASP were stripped and reprobed with anti-β-actin antibody. Blots were cropped following the corresponding molecular weight of the protein of interest.Western blot samples were prepared by immediately adding Laemmli buffer to washed platelets inside the cuvettes and boiled at 95 °C for 10 min. Platelet proteins were separated by electrophoresis using 8% SDS-PAGE gels then transferred to polyvinylidene difluoride 8/ml in presence of LiCl (1 mM), inhibiting the degradation of InsP1 into myo-inositol. Washed platelets were activated by EB in the absence or presence of effectors, under stirring conditions. Platelets were lysed after 5 min of stimulation then centrifuged at 16,000 x g for 10 min at 4 °C. Platelets lysates were incubated with InsP1- HRP conjugate and anti- InsP1 monoclonal antibody for 3 h.The production of inositol triphosphate (InsP3) was measured by the level of the InsP1 accumulated in washed platelets using the IP-One ELISA kit according to the manufacturer’s instructions. Human washed platelets adjusted to 3 × 108/ml in Hepes buffer. Washed platelets were then pre-incubated with a Ca2+ indicator dye, fluo-3 acetoxymethyl (AM) esters (5 μM) for 30 min at 37 °C. Intracellular Ca2+-release was monitored for 2 min by flow cytometry after stimulation with EB without additional supplementation of extracellular Ca2+.Human platelets were prepared as already mentioned above and adjusted to 3 × 10P < 0.05 was considered as significant.Experiments were performed at least three times with at least three different healthy donors. The data were presented as mean ± standard deviation (S.D.). Statistical analysis was performed using GraphPad Prism 7 for Windows . The 2-tailed Student’s t-test was used for comparison of two groups, one-way and two-way ANOVA and Tukey’s multiple comparison tests were used, when appropriate, for comparison of more than two groups. Echis carinatus sochureki produced variable effects on platelets and prompted us to modify the purification procedure , with the SFK inhibitor PP2 and its inactive analogue PP3. EB-induced platelet aggregation was strongly inhibited only in the presence of PP2 , which showed severe reduction of the major fibrinogen receptor αIIbß3 and which has been studied previously [IIbβ3 inhibitor tirofiban on Syk activation with platelets from healthy controls. EB-induced Syk phosphorylation in platelets pre-incubated with tirofiban was not different when compared to non-treated control platelets. Also, the combination of iloprost and tirofiban did not differ in the phosphorylation pattern of Syk compared to platelets pre-incubated with iloprost alone  by functional studies, immunoblotting and phosphoproteomic analysis using iloprost or riociguat –54. HereThen we also used lower concentrations of iloprost (1 nM) and riociguat (10 μM), which partially inhibit EB-induced platelet aggregation, to test if the observed protein phosphorylation effects are still present compared to 2 nM iloprost and 20 μM riociguat, respectively. For 1 nM iloprost  on EB-simulated platelet aggregation and Syk activation prompted us to study also a classical mechanism of Syk activation in human platelets, the convulxin/GPVI pathway –12. For 2+-release upon platelet activation with EB, which was abolished by PRT-318. However, iloprost and riociguat showed only partial inhibition of EB-induced Ca2+-release. In addition, EB-induced Ca2+-release showed to be independent from the integrin αIIb β3 outside-in signaling as tirofiban had no significant effect  as marker of EB-mediated InsP3 production as reported . We deteIn this study we demonstrated that cAMP/PKA and cGMP/PKG pathways cause dichotomous regulation of GPIbα-mediated Syk stimulation and activation of human platelets. To ensure selective GPIbα-activation the specific GPIbα-ligand echicetin was used as multimer complex, coated on polystyrene beads (EB), leading to integrin αIIbβ3-dependent aggregation of human platelets –41. EchiEB induced platelet Syk phosphorylation and aggregation, which requires SFKs as these effects are prevented by the SFK inhibitors PP2 and dasatinib. Syk activation by EB in human platelets as assessed by Y352 and Y525/526 phosphorylation, was rapid but transient, indicating substantial dephosphorylation. Interestingly, dephosphorylation of murine Syk at Y346 (~ Y352 in the human kinase) by the TULA-2 protein-tyrosine phosphatase suppressed its activation in murine platelets .As reported by us recently, EB strongly activated murine platelets, expressing only FcRγ-chain but not FcRγIIA, which required GPIbα . Our preAltogether, the data indicate that EB activation of the human platelet GPIb-complex stimulates Syk phosphorylation via an SFK-dependent mechanism, which is antagonized by EM. SFK-dependent Syk activation involves direct Syk Y352 phosphorylation and may also involve the platelet ITAM protein FcRγ-chain.To validate a functional role of Syk for GPIbα-signaling in human platelets, we tested two different Syk inhibitors, OXSI-2 and PRT-318. These have been described as Syk and platelet function inhibitors in studies of convulxin/GPVI signaling –50. PRT-OXSI-2 (2 μM) and PRT-318 (1 μM) abolished EB-induced aggregation and strongly inhibited EB–induced Syk Y525/526, but not Syk Y352, phosphorylation. These inhibitors also prevented EB-induced phosphorylation of the direct Syk substrate (PLCγ2 at Y759) and a further downstream effector, Akt S473. Syk Y525/526 phosphorylation, primarily due to autophosphorylation , 24, is Based on the inhibitory effects of the Syk inhibitors, EB-induced Akt phosphorylation requires Syk and therefore appears to be downstream of Syk activity. However, Akt is certainly not an immediate Syk target/direct substrate but is most likely phosphorylated in response to additional adapter proteins affecting PI3K , which iA recent paper reported that Syk activity is dispensable for platelet GPIb-IX-V signaling induced by ristocetin/vWF  by showi2) for a full response, with certain differences [12 and P2Y1) and the TxA2 receptor were blocked. In contrast, Syk phosphorylation was not at all (Y352) or only partially (Y525/Y526) reduced, whereas PLCγ2 Y759 and Akt S473 phosphorylation was strongly reduced. We have no clear explanation for the partial inhibition of Syk 526/526 phosphorylation by the secondary mediators except to speculate that this site may also be affected by pathways other than SFKs. This has also been proposed for PLCγ2 and its phosphorylation [The receptors GPVI and CLEC-2 activate platelets by a Syk-dependent mechanism and require the release of secondary mediators -dependent Syk stimulation [IIbβ3 for EB-induced signaling, we compared platelets from normal controls and αIIbβ3-deficient platelets from a patient with Glanzmann thrombasthenia. There was no difference between normal and αIIbβ3-deficient platelets with respect to EB-induced Syk activation as indicated by Y352 and Y525/Y526 phosphorylation. Similarly, the αIIbβ3 inhibitor tirofiban did not affect EB-induced Syk tyrosine phosphorylation and phosphorylation of the Syk substrate PLCγ2 at Y759. These data show that EB/GPIb-mediated phosphorylation and activation of Syk is integrin αIIbβ3-independent.In addition to ADP and TxAmulation , 60. To Platelet functions are tightly regulated by a network of intracellular pathways consisting of protein kinases/protein phosphatases and their substrates , 8, 61, 2+-release and its functional roles remained unclear until now [2+-release in human platelets, which was completely GPIbα-dependent (response abolished by EM), Syk-dependent (abolished by PRT-318) and integrin αIIbβ3-independent (unaffected by tirofiban). However, iloprost (cAMP) and riociguat (cGMP) pre-treatment only partially inhibited EB-stimulated InsP1 increase and Ca2+-release. These only partial inhibitory effects differ from the known strong PKA/PKG-mediated inhibition of the InsP3/Ca2+ response by platelet GPCR  activation, which occurs at several sites including GPCR signaling, PLCβ, and the InsP3 receptor or its associated protein IRAG [2-stimulated Ca2+-release from intracellular stores in human platelets was essentially abolished by the PKA and PKG pathway [2+ response but perhaps stronger at sites downstream of InsP3 production and Ca2+ elevation. Interestingly, earlier studies using PLCγ2-deficient murine platelets and vWF/botrocetin suggested that PLCγ2 is not required [2+-release were found to be dependent on the secondary mediators ADP and TxA2, different from the initial Syk phosphorylation/activation. It is very likely that important functional responses, such as Ca2+-release from intracellular stores, are heterogeneous and controlled by several pathways and proteins. A possible hotspot for the integration of various in-going Syk signals and out-going responses is the membrane protein LAT, an important Syk substrate and component of ITAM/Syk signaling in immune cells and also in platelets [Syk-mediated PLCγ2 activation and InsP3 and 1,2-diacylglycerol (DAG) generation are essential components of the GPVI-pathway in human and murine platelet, and most GPVI/ITAM-effects are thought be mediated by Syk , 13, 18,ntil now , 35, 65.ein IRAG , 66, 67.latelets , 68.Interestingly, we observed similar effects of cAMP/PKA and cGMP/PKG pathway stimulation on GPVI-mediated Syk phosphorylation. Whereas convulxin-stimulated platelet aggregation was completely inhibited by iloprost (cAMP) and riociguat (cGMP), convulxin-induced Syk phosphorylation (Y352 and Y525/526) was not inhibited, but instead clearly enhanced/prolonged (hyperphosphorylation) by iloprost or riociguat pretreatment. Convulxin-stimulated PLCγ2 Y759 phosphorylation was also enhanced by the cAMP/cGMP pathways, whereas Akt S473 phosphorylation was inhibited. In line with this observation, stimulation of CLEC-2  on activated mouse platelets was shown to be only weakly inhibited by the cAMP, and not by the cGMP pathway . Our cvxPresently, the mechanism(s) for the PKA/PKG-mediated increase of Syk Y352 and Y525/526 phosphorylation (hyperphosphorylation) and kinase activity is a topic of further investigation in our laboratory. Interestingly, a possibly related “Syk Y-hyperphosphorylation” has been observed in response to PKC inhibitors . Recentl2. Both PKA/PKG pathways block platelet activation by ADP, TxA2, thrombin [In contrast to Syk tyrosine phosphorylation, further downstream effects such as PI3K activation/Akt and aggregation in response to EB or convulxin were strongly inhibited by the PKA/PKG pathways or by blocking the secondary mediators ADP/TXAthrombin , 9, 67, thrombin , 73. PKAthrombin , 9, 61. Our data establish that selective activation of GPIbα in human platelets by using echicetin-beads results in SFK-dependent Syk activation, subsequent Syk-mediated signaling, and ultimately integrin-dependent aggregation. In contrast to others  we obtaiSyk pathway components display a variable dependency on secondary mediators  and variable regulation by cAMP/cGMP inhibitory pathways, revealing two distinct directions of GPIbα−/Syk signaling. The initial GPIbα-caused Syk phosphorylation, activation and direct substrate phosphorylation is independent of the integrin αIIbβ3 and secondary mediators. Then, two distinct platelet inhibitory mediators, cAMP/PKA and cGMP/PKG, completely block one part of GPIbα−/GPVI-stimulated Syk signaling responses , but increase the initial receptor-induced Syk activation and phosphorylation of two important direct Syk substrates, PLCγ2 and LAT. The observation that cAMP and cGMP abolish GPIbα-mediated aggregation but not Syk activation under our conditions suggests that the inhibitory PKA/PKG effects occur downstream of Syk activation, not at the level of Syk activation/Syk activity. Our results indicate the presence of distinct Syk signaling/effector systems, which differ in their interaction with other signaling pathways. The physiological and pathophysiological significance of these different Syk effector systems needs further investigation. This is clearly warranted in the light of the functional importance of Syk in platelets and immune cells, and in the light of Syk and related tyrosine protein kinases as important pharmacological target.Additional file 1:Figure S1. Echicetin purification and qualitative validation. Echicetin was isolated from the snake venom of Echis carinatus sochureki by affinity chromatography followed by anion exchange chromatography using DEAE column. a Elution was performed with a gradient of NaCl, from 0 to 1 M at a flow rate of 1 ml/min. Two main peaks were separately eluted (P1 and P2) at around 62.5 and 167 mM, respectively. b Samples from P1 and P2 were analyzed by silver staining under non-reducing (NR) and reducing conditions (R) in a 15% SDS-gel. Bands detected under reducing conditions were cut from the gel, digested using trypsin, and analyzed by MS-analysis. The upper and lower bands of peak 1 were identified under the Uniprot IDs: P81017 (echicetin α-subunit) and P81996 (echicetin β-subunit), respectively. However, the upper band of peak 2 was detected as a sequence not related to Echis carinatus sochureki species, and the lower band was identified under the Uniprot ID: P81996 (echicetin β-subunit). For all experiments, echicetin from peak 1 was used. c Representative aggregation curves of human washed platelets (WP), which were stimulated under stirring conditions with EB or BSA-coated beads (as negative control). WP were pre-incubated with echicetin (EM)  or with tirofiban  prior to stimulation with EB. d Corresponding quantitative data of platelet aggregation expressed as maximum percentage of light transmission. Results are shown as means ± S.D. of 3 independent experiments with platelets from 3 healthy donors (****p < 0.0001). (PPTX 520 kb)Additional file 2:Figure S2. Echicetin monomer does not affect aggregation induced by GPVI or by G-protein coupled receptors. Washed platelets were pre-incubated with 25 μg/ml echicetin monomer for 3 min before stimulating with a collagen (3 μg/ml), c ADP (1.75 μM), e TxA2 (60 nM), g TRAP6 (5 μM) and i-k thrombin (0.1 or 0.03 U/ml). Quantitative data are presented as means ± SD for b collagen, d ADP, f TxA2, h TRAP-6, j,l thrombin. Data are presented from 3 different experiments with platelets from 3 different healthy donors; ns: not significant. (PPTX 174 kb)Additional file 3:Figure S3. EB-induced platelet aggregation is dependent on src family kinases (SFKs). Washed human platelets (WP) were pre-incubated for 5 min with the SFK inhibitor, PP2 (10 μM) or with its inactive analogue, PP3 (10 μM) prior to stimulation with EB. a Representative curves of the effect of PP2 and PP3 on platelet aggregation and b the corresponding quantification are shown as means ± S.D. Data are from at least 3 independent experiments with platelets from at least 3 healthy donors; ns: not significant, ****p < 0.0001. (PPTX 70 kb)Additional file 4:Figure S4. Syk inhibitors diminish EB-induced general tyrosine phosphorylation and vWF/ristocetin-induced platelet aggregation and phosphorylation of Syk 525/526, LAT Y191 and Akt S473. a Washed human platelets (WP) were pre-incubated with 2 different Syk inhibitors, OXSI-2 (2 μM) and PRT-318 (1 μM) for 5 min prior to stimulation with EB. Aggregation was stopped after 1, 2 or 5 min using Laemmli buffer. General tyrosine phosphorylation was analyzed by western blot using a pan-phosphotyrosine antibody. b WP were pre-incubated for 5 min with vehicle control or PRT-318 (1 μM) in the presence or absence of 1.25 μg/ml tirofiban prior to stimulation with 2.5 μg/ml human vWF plus 0.5 mg/ml ristocetin. Representative curves show the effect of PRT-318 on vWF/R-induced platelet aggregation. Incubation of WP with tirofiban was used to dissect between vWF-mediated platelet agglutination and integrin αIIbβ3-dependent aggregation. c The corresponding quantification demonstrates the effect of PRT-318 on the residual platelet aggregation response  induced by vWF/risotcetin. Results are calculated from 4 different experiments with platelets from 4 healthy volunteers and data are presented as means ± S.D. **p < 0.01, ***p < 0.001, ****p < 0.0001. d, e Platelet aggregation was stopped after 2 min by addition of Laemmli buffer to analyze Syk Y525/526, Y352 and LAT Y191 and aggregation was stopped after 5 min for the analysis of Akt S473 by immunoblotting. Western blots are representative for at least 3 independent experiments from at least 3 healthy volunteers. (PPTX 380 kb)Additional file 5:Figure S5. Iloprost and riociguat inhibit EB-induced platelet aggregation in a dose-dependent manner and induce stable VASP S157 and S239 phosphorylation. Representative curves of the effect of increased concentrations of a iloprost and b riociguat on EB-induced platelet aggregation. c Washed human platelets (WP) were pre-incubated for 3 and 2 min with 2 nM iloprost and 20 μM riociguat, respectively prior to stimulation with EB. VASP phosphorylation at S239  was analyzed by immunoblotting. VASP S157 phosphorylation at S157  is visible here by the well-established pS157-dependent shift from the 46 kDa to 50 kDa form of VASP in SDS-PAGE. The complete shift of the VASP to the 50 kDa form indicates near stoichiometric VASP S157 phosphorylation by iloprost-activated PKA. Quantification VASP S239 is represented as ratio compared to the loading control β-actin. Data are presented of 3 different experiments with platelets from 3 healthy volunteers as means ± S.D. ****p < 0.0001. (PPTX 292 kb)Additional file 6:Figure S6. Syk and PLCγ2 tyrosine phosphorylation is also increased and prolonged at 1 nM iloprost. Washed human platelets (WP) were pre-incubated 3 min at 37 °C in the presence or absence of iloprost (1 nM) prior to stimulation with EB. Phosphorylation of a Syk Y525/526 and Y352, d PLCγ2 Y759 and Akt S473 was analyzed by immunoblotting in a time dependent manner. b, c, e, f Quantification of the phosphorylated proteins are represented as ratio compared to the corresponding total protein. Data are shown of 3 different experiments with platelets from 3 healthy volunteers as means ± S.D. ns: not significant, *p < 0.05, **p < 0.01, ****p < 0.0001. (PPTX 1770 kb)Additional file 7:Figure S7. Syk and PLCγ2 tyrosine phosphorylation is also increased and prolonged at 10 μM riociguat. Washed human platelets (WP) were pre-incubated for 5 min at 37 °C with vehicle control (DMSO) and 10 μM riociguat, respectively prior to stimulation with EB. Phosphorylation of a Syk Y525/526 and Y352, d PLCγ2 Y759 and Akt S473 were analyzed by immunoblotting in a time dependent manner. b, c, e, f Quantification of the phosphorylated proteins are represented as ratio compared to the corresponding total protein. Data are shown of 3 different experiments with platelets from 3 healthy volunteers as means ± S.D. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. . (PPTX 358 kb)Additional file 8:Figure S8. Iloprost and riociguat inhibit convulxin-induced platelet aggregation but not Syk activation. Washed human platelets (WP) were pre-incubated with iloprost  or riociguat  prior to stimulation with 50 ng/ml convulxin. a Representative aggregation curves and the corresponding quantitative data are shown in b. c Aggregation was stopped after 1, 2 or 5 min using Laemmli buffer. Syk Y525/526 and Y352 were analyzed by western blot. d, e Quantification of the phosphorylated proteins is presented as ratio compared to the total Syk protein. Results are representative of at least 3 different experiments with platelets from at least 3 healthy volunteers, data are presented as means ± S.D. ns: not significant, ****p < 0.0001. (PPTX 181 kb)


Here, Leskoske et al. studied how TORC2 activity is modulated in response to changes in the status of the cell envelope. They demonstrate that TORC2 subunit Avo2 is a direct target of Slt2, the MAPK of the cell wall integrity pathway, and their findings provide new insights into TORC2 function and regulation. Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation. Cellular homeostasis requires careful coordination of metabolic growth processes and adaptive stress responses. Multiple signaling networks coordinate expansion at the cell boundary with the accumulation of intracellular mass and progression through the cell cycle and also play critical roles in responding to environmental fluctuations that compromise the integrity of the plasma membrane (PM) and, in yeast, the cell wall .Like all eukaryotes, yeast contains two evolutionarily conserved multicomponent protein kinase complexes in which a TOR (target of rapamycin) polypeptide is the catalytic subunit: TOR complex 1 (TORC1) and TORC2. TORC1 and TORC2 are essential regulators of growth and homeostasis but control different aspects of yeast cell physiology . TORC2 iConditions that perturb the PM stimulate TORC2-mediated phosphorylation of Ypk1, including sphingolipid depletion , hypotonIn addition, however, other pathways that sense PM or cell wall stress may exert some of their effects via regulation of TORC2 function. In this regard, two mitogen-activated protein kinase (MAPK) pathways in yeast sense and respond to stresses that challenge the cell envelope: the high-osmolarity glycerol (HOG) pathway, whose terminal MAPK is Hog1 , and theSignificant cross-talk exists between the HOG and CWI pathways  and betwIn this study, we sought to establish whether the CWI MAPK pathway directly modulates TORC2–Ypk1 signaling, given the cumulative evidence for the interconnections between them. As documented here, we found that activation of the CWI pathway MAPK results in direct Slt2-mediated phosphorylation of TORC2 subunits and that these modifications down-regulate TORC2–Ypk1 signaling. By pinpointing the molecular basis of this negative regulation, our results provide new mechanistic insight into how TORC2 senses cell envelope stress.Saccharomyces Genome Database, https://www.yeastgenome.org) have detected in vivo phosphorylation of -SP- and -TP- sites in four of the six TORC2 subunits   , which iive Slt2 B. Overexive Slt2 C did notive Slt2 B.slt2Δ and hog1Δ mutants. We found that the Pkc1*-induced mobility shift was markedly reduced in slt2Δ cells but not in hog1Δ cells (9A) in which all nine sites were mutated to Ala. Indeed, absence of these sites prevented Pkc1*-induced hyperphosphorylation of Avo2 , purified in the same mannerA mutant E.Likewise, Avo3(1–100), an N-terminal fragment containing six of the 11 -SP- and -TP- sites in Avo3 (five of which have been detected as phosphorylated in vivo), was robustly phosphorylated in vitro by wild-type Slt2 but not by the catalytically deficient Slt2 mutant, and incorporation was markedly reduced by mutation of the six sites to Ala F. Moreov9A mutant, we also generated a phosphomimetic allele, Avo29E. First, we found that all three proteins were expressed at comparable steady-state levels , that is fully active in the absence of TORC2 phosphorylation (Unlike Avo3 (1430 residues), which is essential for TORC2 function because it has an important structural role , Avo2 ise levels A; thus, e levels . Furthermyriocin B, suggesrylation . These f9E-expressing cells , myriocin treatment failed to stimulate TORC2 phosphorylation of Ypk1 in the cells expressing both Avo29E and Pil13D  Kss1 MAPK  isoforms, concomitant with the appearance of activated Slt2 A, as wele mutant C. We exass1 MAPK D. Howeve mutants C. Most rIt has been reported that Pkc1 is a substrate of TORC2 ; hence, Saccharomyces cerevisiae kinome, we found that, like a tor2ts mutant  that was accompanied by hyperphosphorylation of Avo2 , indicating that, like activation of the CWI MAPK pathway, activation of the HOG MAPK pathway down-regulates TORC2 function and does so via a similar mechanism.Sln1 is a histidine kinase similar to those in bacterial two-component signaling systems, and its inactivation, as occurs during hyperosmotic shock, leads to activation of the Hog1 MAPK . In a gls mutant , when a To confirm this conclusion and inactivate Sln1 independently of any external temperature or hypertonic stress, we constructed a strain expressing the plant F-box protein Tir1 and an allele of Sln1 tagged with a modified auxin-inducible degron (AID*), which should cause Sln1 to be degraded in an SCF-mediated manner upon auxin addition . Adding hog1Δ mutant or a slt2Δ mutant and was not further enhanced in a hog1Δ slt2Δ double mutant  was not sufficient to down-regulate TORC2-mediated Ypk1 phosphorylation . Thus, in contrast to the Slt2-dependent down-regulation of TORC2-mediated Ypk1 phosphorylation that occurs upon prolonged inactivation of Sln1, the loss of the TORC2-dependent Ypk1 isoforms caused by exposure to 1 M sorbitol occurs in a MAPK-independent manner. Possibly, the hypertonic shock caused by 1 M sorbitol activates an as yet to be identified phosphatase. In any event, both acute and chronic stimulation of the HOG pathway leads to down-regulation of TORC2–Ypk1 signaling, albeit by different mechanisms.Interestingly, we documented previously that when the HOG pathway is activated by exposure of cells to 1 M sorbitol, a cell-impermeable osmolyte that robustly but transiently activates Hog1 , there i1Δ cells . AlthougStudies of various integral and peripheral PM proteins in yeast have shown that different components often reside in discrete microdomains . Many pe9A, or Avo29E (each tagged at its C terminus with mNeon Green [mNG])  and (2) that both Avo2-mNG and Pil1-RFP displayed a punctate distribution at the PM and that these puncta were often, but not always, congruent . The same was observed for Avo29A-mNG and Pil1-RFP   from itsn [mNG])  from itsongruent A, left, Pil1-RFP A, middlerominent A, right,nent . However, quantification of the immunoprecipitates showed that, compared with the amount of Avo2 or Avo29A bound to TORC2, the amount of Avo29E was always reduced by 30%–40%. Thus, in agreement with the observations made by fluorescence imaging, these results suggest that MAPK phosphorylation of Avo2 promotes its dissociation from TORC2.To confirm that conclusion for TORC2 specifically by an independent biochemical approach, we immuno-isolated TORC2 (in which the Avo3 subunit carried a 3xFlag epitope tag) from the cells expressing Avo2, Avo2r Avo29E F. LikewiIt has been unclear whether TORC2 adjusts its activity by serving itself as a direct sensor of perturbations to the cell envelope or responding to inputs from other stress-sensing pathways (or both). We demonstrated here, for the first time, that Slt2/Mpk1  , the MAPIn any event, the control circuit that we discovered makes physiological sense because under conditions that interfere with cell wall synthesis or damaged cell wall structure, activation of the CWI pathway will exert feedback that will negatively regulate the growth-promoting functions of TORC2 G. For yeS. cerevisiae and its sensu stricto relatives to divergent yeast species  to filamentous fungi  —but is not conserved in multicellular organisms that lack a cell wall. In this same regard, genetic evidence indicates that the Slt2 ortholog (Pmk1) in fission yeast (Schizosaccharomyces pombe) has a role in negatively regulating TORC2-mediated activation of the fission yeast Ypk1 ortholog (Gad8)  , providiOur results show that Avo2 is not only an important site of Slt2-mediated phosphorylation but also necessary for optimal TORC2 activity, especially under stressful conditions. However, the function of Avo2 in TORC2 is not currently known. Within its N-terminal half, Avo2 contains at least five ankyrin repeats , which a2-binding F-BAR domain-containing proteins Pil1 and Lsp1  and do so via canonical so-called PxIxIT motifs located very near their C termini (673PNIYIQ678 in Slm1 and 640PEFYIE645 in Slm2). Hence, it is possible that the calcineurin bound to Slm1/2 acts to remove the inhibitory phosphorylations present on Avo2 and other TORC2 subunits, thereby alleviating this negative regulation. Consistent with this model, we found that Avo29E, an allele that mimics the permanently phosphorylated state and hence would be immune to calcineurin action, was inhibitory to optimal TORC2 function in vivo, exhibited enhanced dissociation from PM sites, and reduced association with TORC2. Thus, based on the behavior of this phosphomimetic allele, a primary effect of Slt2-mediated phosphorylation is to impede its integration into TORC2 and interfere with Avo2-mediated association of TORC2 with the cell cortex. Whether Slt2 phosphorylation of Avo3 (or other TORC2 subunits) further promotes this dissociation and other ramifications of this model provide fertile ground for further study.Hence, the findings that we describe here suggest an obverse model; namely, that the role of Avo2  is to act as an adaptor that, through its association with Slm1/Slm2, is responsible for the dynamic docking of TORC2 at eisosomes. What, then, is the function of the Avo2–Slm1/2 interaction in promoting TORC2 function? As demonstrated by multiple groups , Slm1/2 Finally, what we reveal here is one molecular mechanism by which a stress signal restrains a growth-promoting signal. As we document here for yeast, it seems likely that MAPK pathways activated by stress will impede TORC2 function in mammalian cells in a similar manner because continued growth and response to stress are invariably incompatible cellular states.S. cerevisiae strains used in this work were all constructed using conventional yeast genetic methods  (Unless indicated otherwise in  methods . Unless  methods  containi6 [GEV]) , by addiEscherichia coli strain DH5α  diluted 1:1 with PBS or TBS, and then probed by addition of the appropriate primary antibody at the indicated dilution: mouse anti-myc mAb 9E10 , mouse anti-Flag M2 mAb , mouse anti-HA.11 mAb , rabbit polyclonal anti-RFP , rabbit polyclonal anti-tRFP , or rabbit polyclonal anti-Pgk1  or pKL64 (PGAL1-Slt2KD-TAP) was grown to mid-exponential phase at 30°C. Expression was induced by addition of 20 µM β-estradiol followed by incubation for 3 h. The cells were harvested, washed once in ice-cold TAP-B buffer containing phosphatase and protease inhibitors , resuspended in 4 mL of TAP-B buffer, and flash-frozen as droplets in liquid N2. The cells were ruptured cryogenically in a Mixer Mill MM301 (Retsch). The resulting lysate was thawed on ice, diluted with 8 mL of TAP-B buffer, and clarified by centrifugation at 15,000g for 20 min. The resulting clarified extract was subjected to centrifugation at 78,000g for 1 h, and the resulting supernatant fraction was adjusted to a final concentration of 0.15% NP-40 using an appropriate volume of a 10% NP-40 stock. Slt2-TAP in the detergent-containing soluble fraction was captured by binding to IgG-agarose resin . After washing the resin extensively with TAP wash buffer , it was washed with Protease 3C buffer . To elute the bound Slt2-TAP, the washed resin was resuspended in 500 µL of Protease 3C buffer containing 60 U of PreScission protease . After incubation for 5 h at 4°C, the resin was removed by centrifugation, and the Protease 3C in the resulting supernatant faction was removed by incubation with a slurry of glutathione-Sepharose beads  followed by centrifugation. The affinity-purified Slt2 in the resulting final supernatant solution was diluted into kinase assay buffer .To purify activated Slt2, 1 L each of yeast strain CGA84 cotransformed with PE. coli BL21(DE3)pLysS. One liter of cultures was grown at 30°C to mid-exponential phase and then induced with 0.5 mM IPTG for 3 h at 30°C. The bacterial cells were harvested by centrifugation and lysed, and the GST fusion protein of interest was adsorbed to glutathione-agarose beads , which were washed and resuspended in kinase assay buffer. The same volume of either affinity-purified Slt2WT or Slt2KD was added to the resuspended beads or an equivalent volume of kinase assay buffer, and reaction was initiated by the addition of 2 µCi of [γ-32P]ATP and unlabeled ATP to 100 µM final concentration. After incubation for 30 min at 30°C, reactions were terminated by addition of 5× SDS-PAGE sample buffer followed by boiling for 10 min. The resulting reaction products were resolved by SDS-PAGE and analyzed by Coomassie blue staining and autoradiography using a phosphorimager .Substrates to be tested were purified as recombinant proteins by fusion to the C terminus of GST in appropriate pGEX vectors and expressed in 9A-mKate (YFR626), or Avo29E-mKate (YFR628) was grown in YPD to mid-exponential phase, harvested by centrifugation, and frozen in liquid nitrogen. Cells pellets were resuspended in 500 µL of 2× TNEG buffer . Glass beads were added to the meniscus of the cell suspension, and lysis was achieved by vigorous vortex mixing for 3 min. After lysis, 2× vol of 1× TNEG buffer was added, and then the lysate was clarified by centrifugation at 1000g for 5 min at 4°C followed by a second centrifugation at 2000g for 10 min. TORC2 complexes marked by the tightly bound Avo3-3xFlag subunit were collected by immunoabsorption to 40 µL of mouse anti-Flag affinity gel (Biotool) equilibrated in 1× TNEG buffer. The resin was washed four times with 1 mL of 1× TNEG buffer and resuspended in 2× urea buffer.One-hundred milliliters of cultures of yeast strains expressing Avo3-3xFlag Tor2-mNG-3xHA and either Avo2-mKate (YFR624), Avo2max 506 nm; emission λmax 517), cell samples were excited with an argon laser at 488 nm at 2.3% power (100 mW), and emission was captured in a 495- to 550-nm window using a bandpass filter; for Pil1 tagged with RFP/DsRed , excitation was at 561 nm at 2.3% power (100 mW), and emission was monitored in a 570- to 620-nm window using a different bandpass filter. Images  were analyzed using Fiji (http://www.R-project.org).Fluorescence microscopy was performed using an Elyra PS.1 structured illumination microscope (Carl Zeiss AG) equipped with a 100× plan-apo 1.46NA TIRF objective, a main focus drive of the AxioObserver Z1 stand, and a WSB PiezoDrive 08 controlled by Zen, and images were recorded using a 512 × 512 (100-nm × 100-nm pixel size) EM-CCD camera (Andor Technology). To visualize Avo2 or Tor2 tagged with mNG (excitation λing Fiji . To avoiing Fiji . For quaing Fiji . To traiing Fiji . Prior ting Fiji . To avoi