The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC. However, optimizing the efficient expansion and differentiation of these cells still requires further understanding, especially of the signaling pathways responsible for regulating hESC. Several pathways have been implicated in hESC self-renewal, including transforming growth factor-β/Activin-A/Nodal et. al. 2006 Human ESCs are a powerful tool for drug screening, studying early lineage differentiation FGFs execute their biological actions by activating cell surface fibroblast growth factor receptors (FGFRs) The large scale analysis of cellular phosphorylation by liquid chromatography-mass spectrometry (LC-MS) is challenging due to the low stoichiometry of phosphorylation, causing phosphopeptides to remain largely undetected in the overwhelming background of non-modified peptides. Several of these large scale proteomic methods have been applied to profile phosphorylation in hESC To understand the broader implications of FGF signaling in hESC, we adopted a large-scale, targeted, phosphoproteomics approach to investigate tyrosine phosphorylation events following FGF-2 stimulation. Using a peptide-centered immuno-affinity purification strategy To specifically investigate the effects of activating FGFR, exogenous FGF-2 was added to hESC that were deprived of FGF-2 for 5 days (1 day ∼ 1 population doubling). Activation of the hESC was validated by Western blotting using two antibodies specific for the phosphorylation of proteins downstream of the FGFR. After 15 min of stimulation, a rapid upregulation of both phosphorylated ERK1/2 (ph-ERK1/2) and AKT (ph-AKT) was observed .A targeted phosphoproteomics experiment was subsequently carried out to profile the global tyrosine phosphorylation events post FGF-2 stimulation using an anti-pY specific antibody to enrich for tyrosine phosphorylated peptides A comparison of our hESC dataset with known pY site locations obtained from Phospho.ELM (version 8.2) and Rikova et al. In order to accurately quantify the tyrosine phosphorylation events, we next applied a combined approach of pY-peptide immunoprecipitation (IP) and stable isotope labeling. Using this combined approach, we have generated a more precise and quantifiable tyrosine phosphorylation profiles Interestingly, various substrates of the Src kinase family were also found within these clusters, namely Shank2, F11R, PKP2, TJP2, CDK5 , and CTTN, OCLN, FAK, PKP3, PXN, and TJP1  see . The SrcTo confirm the increased phosphorylation of the RTKs identified post FGF-2 stimulation, a protein array containing 42 different human RTKs  was used . In the Many cytokines and growth factors have been shown to play a role in maintaining self-renewal of hESCs  demonstrated rapid response (5–15 min) of these downstream effectors to FGF-2 stimulation, hence we selected 0, 5, and 15 min for a more in-depth quantitative study. Stable isotope dimethyl labeling method was selected to be used in conjunction with pY-peptide IP. By performing stable isotope labeling prior to IP, potential variation in the IP or LC-MS step is mitigated as sample handling and analysis is performed simultaneously for all samples. A potential downside is that up to 6 mg of material needs to be isotope labeled, necessitating a cost-effective labeling approach provided by reductive dimethyl labeling. Using the above combination, 316 unique quantifiable tyrosine phosphorylated peptides, occurring as a triplet in the MS data, could be identified and quantified.et. al (2004) As expected, increased tyrosine phosphorylation at multiple sites of FGFRs was observed after FGF-2 stimulation, notably on all four FGFRs. The use of the RTK array confirmed the simultaneous activation of the four FGFRs. A separate study by Livia Apart from FGFRs, several other receptors were found to be rapidly tyrosine phosphorylated following FGF-2 stimulation. These included INSR, IGF1-R, ERBB2, ERBB3, EphA1 and 2, EphB3 and 4, VEGFR2/KDR, and MEGF10. The activation of most of these receptors was confirmed by a human phospho-RTK array. IGF1-R and IR have been shown to play a role in self-renewal of hESC One possible explanation for the induced tyrosine phosphorylation of other RTKs following FGF-2 stimulation could be transactivation Src family kinases (SFKs) represent a group of tyrosine kinases that are strongly activated by, amongst others, RTKs Cortactin has long been known to be a very efficient substrate for SFKs and was found in our screen to exhibit increased tyrosine phosphorylation levels. These phosphorylation events are supposed to create docking sites for proteins containing the SH2 phosphotyrosine binding domain, such as Src family kinases themselves or adaptor proteins Plakophilin-3, Plakophilin-4, Catenin delta-1, F11R, Occludin, KIRREL/Neph1, TJP-1 and TJP-2 are found in tight junctions and an increased tyrosine phosphorylation was detected in our screen. Of these proteins Catenin delta-1, Occludin, TJP-1 and TJP-2 have been described as genuine Src kinase substrates; the phosphorylation of the aforementioned sites has been shown to modulate the regulation of cell-cell adhesions as well as the formation of tight junctions et. al. (2004) suggest a considerable involvement of these kinases and their substrates in hESC in maintenance of undifferentiated hESC.The prevalence of activity of Src family kinases can be seen from both Motif mapping (Motif 1 and 4) and the large number of Src kinase substrates identified. SFKs can also be recruited by receptor mediated phosphorylation and influence the signaling dynamics of the cell. It has been stated that Src activity controls FGFR activation and FGF-2 induced AKT activation is dependent on activation of Src CDK5 was also found to be upregulated upon FGF-2 treatment. CDK5 is an atypical member of the cyclin dependent kinase family, which has rather been found to be involved in the regulation of CNS development Multiple EGF-like domains 10 (MEGF10) was identified in the dataset with an average of 5 fold-change on all the four pY-peptides identified (within 5 min of FGF-2 stimulation). Out of the four pY-peptides identified, two were not reported previously. Additionally, this is the first account for identification of tyrosine phosphorylation of MEGF10 in hESC, but its precise function in hESC is largely unknown. MEGF10 is believed to be the mammalian ortholog of nematode CED-1, a cell surface receptor involved in phagocytosis of dead cells In this study, we have provided a global temporal tyrosine phosphorylation profile of hESC after FGF-2 stimulation. In Human ESC line, HES-3  was obtained from ES Cell International . Briefly, the cells were cultured on Matrigel-coated  tissue culture dishes and supplemented with conditioned medium from MEF. For routine culture, the medium was supplemented with 10 ng/ml of FGF-2 , and medium was changed daily. The cultures were passaged weekly following enzymatic treatment as previously described PhosSTOP  in the presence of protease inhibitors. Protein concentration was determined using Bradford Assay. Total protein lysate of 6 mg per time point were reduced with dithiothreitol (DTT) at a final concentration of 10 mM at 56°C. Subsequently, lysates were alkylated with 55 mM iodoacetamide. Lysates were diluted 6-fold with 100 mM ammonium bicarbonate and digested overnight with trypsin.Cells were lysed on ice in 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, 50 µg/ml DNase, 50 µg/ml RNase, 1 mM sodium orthovanadate and 1X 2 and cyanoborohydride, formaldehyde-D2 and cyanoborohydride and formaldehyde-13C–D2 and cyanoborodeuteride to generate light, intermediate and heavy dimethyl labels, respectively. The light, intermediate and heavy dimethyl labeled samples were mixed in 1∶1∶1 ratio based on total peptide amount, determined by analyzing an aliquot of the labeled samples on a regular LC-MS run and comparing overall peptide signal intensities.Tryptic peptides were desalted using a Sep-Pak C18 column , eluted peptides were lyophilized, and re-suspended in 100 µL of triethylammonium bicarbonate (100 mM). Subsequently, stable isotope dimethyl labeling was performed on the full digested lysate (∼6 mg) as described before Prior to LC-MS analysis, the differentially labeled peptides from the three different digested lysates were mixed, desalted with Sep-Pak C18 column, and lypholized. Labeled peptide mixtures were dissolved in IP buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NOG, and 1x complete mini protease inhibitor cocktail (Roche diagnostics). Agarose-conjugated anti-p-Tyr (pY99) antibodies  (prewashed three times with IP buffer) were added into each peptide mixture and incubated overnight at 4°C with gentle rotation. After incubation, the beads were then washed three times with 1 ml of IP buffer followed by two times with 1 ml of water, all at 4°C. Peptides were eluted with 0.15% TFA and centrifuged at 1500 g for 1 min to separate the antibody beads from the eluate. Eluted peptides were desalted and concentrated on STAGE-tips.tm C18, 5 µm, ; 20 mm × 100 µm ID, packed in-house) at 5 µL/min in 100% solvent A (0.1 M acetic acid in water). Subsequently, peptides were transferred to an analytical column  at ∼100 nL/min in a 2 hour (non-labeled) or 3 hour (stable isotope dimethyl labeled) gradient from 0 to 40% solvent B (0.1 M acetic acid in 8/2 (v/v) acetonitrile/water). The eluent was sprayed via distal coated emitter tips (New Objective), butt-connected to the analytical column. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS/MS. Full scan MS spectra (from m/z 300–1500) were acquired in the Orbitrap with a resolution of 60,000 at m/z 400 after accumulation to target value of 500,000. The three most intense ions at a threshold above 5000 were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35% after accumulation to a target value of 10,000.Nanoflow LC-MS/MS was performed by coupling an Agilent 1100 HPLC system  to a LTQ-Orbitrap mass spectrometer  as described previously 2 spectra were converted to single DTA files using Bioworks 3.3 at default settings. Runs were searched using an in-house licensed MASCOT search engine (Mascot version 2.1.0) software platform  against the Human IPI database version 3.36  or version 3.37  with carbamidomethyl cysteine as a fixed modification. Light, intermediate and heavy dimethylation of peptide N-termini and lysine residues (for labeled samples only), oxidized methionine and phosphorylation of tyrosine, serine and threonine were set as variable modifications. Trypsin was specified as the proteolytic enzyme and up to two missed cleavages were allowed. The mass tolerance of the precursor ion was set to 5 ppm and for fragment ions 0.6 Da. Peptides were assigned to the first protein hit reported by Mascot. The assignment of phosphorylation sites of identified phosphopeptides was performed by the PTM scoring algorithm implemented in MSQuant as described previously All MS2 of the median of all peptides quantified. StatQuant, an in-house developed program Quantification of peptide triplets of which at least one has obtained a Mascot peptide score of 20 was performed using an in-house dimethyl-adapted version of MSQuant Cells were lysed in 1X Cell Lysis Buffer  supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF). Protein concentrations were determined using the DC protein assay . Twenty micrograms of each sample was mixed with Laemmli buffer and boiled for 5 min at 95°C. All samples were subjected to SDS-PAGE and electro-transferred onto PDVF membranes . Membranes were probed using the corresponding primary antibodies at the indicated dilutions. After incubation with the primary antibodies, appropriate peroxidase-conjugated secondary antibodies  or fluorescent secondary antibodies (LI-COR Biosciences) were used to detect the bound antibodies. Protein bands were visualized either using a chemiluminescence detection reagent ECL Plus  or LI-COR ODYSSY imaging system . All antibodies used are shown in Analysis of protein expression using the human phospho-RTK antibody array  was performed according to manufacturer's instructions. Briefly, capture and control antibodies were spotted in duplicates on nitrocellulose membranes and incubated overnight with 300 µg protein lysate. The membrane was then washed extensively with buffer provided and, further incubated with pan anti-phospho-tyrosine antibody conjugated to horseradish peroxidase (HRP). After incubation, arrays were washed and visualized using chemiluminescence ECL Plus (Amersham).et. al. et. al. datasets respectively. Overlap was determined by counting the number of identical sites between the datasets.The hESC phosphoproteome was compared to other datasets by mapping known pY site locations obtained from Phospho.ELM (version 8.2) and Rikova Figure S1Activation of FGF-signaling in hESC. Activation profile of HES-3 cells post-FGF-2 induction, by Western Blotting. Cells were starved of FGF-2 for 5 days and 10 ng/ml of FGF-2 was added at the indicated time.(TIF)Click here for additional data file.Figure S2Comparison of hESC cumulative tyrosine phosphorylated dataset. Overlap of identified phosphotyrosine sites between our dataset (hESC), those deposited in Phospho.ELM (ver. 8.2), and those identified in Rikova et. al. (TIF)Click here for additional data file.Figure S3Effect of Src Kinase inhibitor on undifferentiated hESC. Cells were treated with increase concentrations of Src Kinase inhibitor (SU6656) for 6 PD. (A) Morphology of hESC after treatment with SU6656. Cells treated with 4 µM of SU6656 showed significant reduce in cell number when compared to control (untreated hESC) Scale bar  =  500 µm and 100 µm respectively (B) Cell count of SU6656 treated hESC. (C) Pluripotent marker expression of SU6656 treated hESC using quantitative real time PCR. Data were expressed as mean + SEM and results were from triplicate runs.(TIF)Click here for additional data file.Table S1Summary of antibodies used.(PDF)Click here for additional data file.Table S2List of cumulative phosphopeptides detected from FGF-2 stimulated hESC.(PDF)Click here for additional data file.Table S3Motif assignment for phosphopeptides from the cumulative dataset.(PDF)Click here for additional data file.Table S4List of phosphopeptides from the quantitated dataset.(PDF)Click here for additional data file.Table S5List of phosphopeptides classified into 5 clusters using cluster analysis.(TIF)Click here for additional data file.Text S1Supplementary Materials and Methods.(DOC)Click here for additional data file.


All of the components of Metabolic syndrome (MetS) have been regarded as risk factors for coronary artery disease (CAD). Early detection of CAD in asymptomatic patients with MetS remains a challenge. Cystatin C,which has been proposed as a novel marker of renal dysfunction,is correlated with mortality in CAD, The purpose of the study was to evaluate whether cystatin C is a potential marker of asymptomatic CAD in MetS patients with normal kidney function.A total of 211asymptomatic MetS patients without prior history of CAD patients were included in a cross-sectional study. Patients were divided into MetS with asymptomatic CAD (n = 136) and MetS without CAD (n = 75) groups according to coronary angiograph results. Serum cystatin C levels were measured using particle enhanced immunonephelometric assays. We first assessed whether there is an independent association of cystatin C with the presence and severity of asymptomatic CAD. Then, we investigated the association between cystatin C and other biochemical risk factors for atherosclerosis.P = 0.004). A multiple logistic regression analysis demonstrated cystatin C was independently associated with the presence of asymptomatic CAD . On receiver operating characteristics (ROC) analysis, the area under the curve (AUC) was 0.622 , and cystatin C showed a moderate predictive value. Furthermore, cystatin C was independently correlated with Gensini score , and serum cystatin C levels increased with the increasing of number of disease vessels (P = 0.005). In a multiple stepwise regression analysis, uric acid (UA)(P < 0.001), body mass index (BMI)(P = 0.002), triglyceride(TG)(P = 0.03), estimated glomerular filtration rate (eGFR)(P < 0.001), and fibrinogen(P = 0.001) were independently associated with cystatin C.Serum cystatin C levels in patients with asymptomatic CAD were significantly higher than those without CAD (Serum cystatin C in our study was significantly associated with the presence and severity of asymptomatic CAD in MetS patients with normal kidney function, suggesting that cystatin C is probably more than a marker of glomerular filtration rate. Metabolic syndrome (MetS) is a clinical entity characterized by abdominal obesity, hyperglycaemia, hypertension, and dyslipidaemia. Until now, the pathophysiology of MetS is not well defined . Each ofCystatin C belongs to a family of competitive inhibitors of lysosomal cysteine protease and is synthesized at a constant rate in all nucleated cells . CompareIn addition to the association with atherosclerosis, connections between cystatin C and features of the MetS have also been illustrated by a few recently published studies ,15. In a2 calculated by Cockcroft-Gault formula, serum creatinine > 133umol/L), hepatic dysfunction were excluded from the study. Studies have shown that large doses of glucocorticoids would increase the production of cystatin C [This was a cross-sectional study consisted of 211 asymptomatic MetS patients without prior diagnosed CAD and with normal eGFR who presented for a annual medical evaluation at QiLu Hospital of Shandong University from March 2010 to March 2012. All patients had abnormal ECG findings include the ST segment abnormalities, T wave abnormalities, and other abnormalities indicate myocardium ischemia and referred to a coronary artery angiography for suspected asymptomatic CAD. The patients were divided into MetS with asymptomatic CAD (n = 136) and MetS without asymptomatic CAD (n = 75) groups according to their coronary angiography results. Individuals with angina pectoris or anginal-equivalent symptoms suggestive of CAD, valvular heart disease, malignancy, inflammatory disease, renal dysfunction The diagnostic criteria for MetS were based on the revised Adult Treatment Panel III criteria (revised ATP-III) . Because2.Reduced high-density lipoprotein cholesterol (HDL-C): ①HDL-C <1.03 mmol/L  or < 1.29 mmol/L ; ②Hyperglycaemia: fasting plasma glucose (FPG) ≥ 5.6 mmol/L or antidiabetic medication use; ③Hypertension: systolic blood pressure or diastolic blood pressure(SBP/DBP) ≥ 130/85 mmHg and/or diagnosed hypertension treated with antihypertensive therapy; ④Elevated triglycerides (TG): TG ≥ 1.7 mmol/L or receiving specific treatment for this lipid abnormality; ⑤Body mass index (BMI) ≥25 kg/m(2)Asymptomatic CAD patients were diagnosed according to the Monica criteria : two or 2). Systolic and diastolic blood pressures were measured using standard methods.Demographic characteristics and cardiovascular risk factors were obtained from medical records. The cardiovascular risk was assessed in terms of hypertension (blood pressure ≥140/90 mmHg or taking anti-hypertensive drugs), smoking status (≥20 packs per year), and diabetes mellitus . Hypercholesterolemia . BMI was calculated as weight (kg) divided by the square of the height , low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol(HDL-c), triglycerides (TG), uric acid(UA) and fasting plasma glucose(FPG) using an automatic biochemistry analyzer . Concentrations of serum cystatin C were determined using Roche Cobas 8000 modular analyzer Series  with a particle-enhanced immunonephelometric assay .The laboratory reference ranges for cystatin C was 0.4 to 8.0 mg/L, and the intra-assay and inter-assay coefficients of variation were 0.9 % and 2.9 %, respectively. Fibrinogen was measured using Von Clauss methods on an electromechanical coagulometers . Serum creatinine(sCr) levels were measured using an enzymatic method, and eGFR was estimated with the Cockcroft-Gault formula . ParticiCoronary artery angiography was performed using standard Judkins techniques. Angiographic analysis was conducted by two expert investigators who were blinded to the study protocol. The extent of CAD was described as 0-, 1-, 2-, or 3-vessel disease based on the number of coronary vessels with >50% luminal narrowing. The severity of coronary atherosclerosis was scored based on Gensini scoring .t test was used for normally distributed variables and the Mann–Whitney U-test was used for skewed variables. Pearson and partial correlation analysis were applied to determine factors that correlated with cystatin C, and Spearman correlation analysis was performed to determine factors that correlated with Gensini score. To determine the independent parameters correlated with cystatin C and the Gensini score, the parameters that correlated significantly in the univariate analysis and other parameters that were biologically relevant to cystatin C and the Gensini score were tested using multiple stepwise regression analysis. A logistic regression analysis was performed to assess the association between the presence of CAD and cystatin C levels. Age, gender, BMI, smoking state, hypertension, diabetes, lipid profile, and eGFR were considered to be potential confounders and were adjusted in the different models. In addition, the receiver operating characteristics (ROC) curve was used to determine the cystatin C cut-off for predicting asymptomatic CAD. All statistical analyses were performed using SPSS 16.0 software for Windows . A two-tailed P-value < 0.05 was set as the level of significance.Continuous variables with normal distribution were expressed as the mean ± SD, while percentages were used to express categorical variables. Variables such as TG, HDL-C, Gensini score that were not normally distributed, as determined by the Kolmogorov–Smirnov test, were logarithmically transformed before analysis and expressed as medians with inter quartile range. The chi-square test was used to compare the categorical variables of subjects with and without CAD. An unpaired student’s P = 0.011).The rates of hypertension and diabetes mellitus in patients with asymptomatic CAD were higher than in patients without CAD, and asymptomatic CAD patients showed an increase of FPG (P = 0.011). The fibrinogen concentrations were significantly higher in patients with asymptomatic CAD than in those without CAD (P = 0.012). However, there was no difference in sCr, eGFR and lipid profiles between patients with asymptomatic CAD and without CAD. Among the patients with MetS, those with asymptomatic CAD had significantly higher serum cystatin C levels than those without CAD , fibrinogen , FPG  and smoking  were independent predictors of the presence of the asymptomatic CAD.Multiple logistic regression analysis was performed using the presence of the asymptomatic CAD as a dependent variable Table . The anaP = 0.003). A serum cystatin C level of ≥0.825 mg/L predicted the presence of CAD with a sensitivity of 71.3% and specificity of 54.7%  of 0.622 . After further adjustment for age, sex and eGFR, the positive correlation remained significant between cystatin C and UA, fibrinogen . A positive correlation was also observed between cystatin C and TG and BMI , BMI , TG , eGFR , and fibrinogen  were independently associated with serum cystatin C.We next investigated the relationship between serum cystatin C levels and clinical biochemical parameters. The Pearson correlation analysis demonstrated that serum cystatin C was positively correlated with age, UA, sCr, and fibrinogen and negatively correlated with eGFR .The cystatin C level in the group N = 0 was significantly lower than those in the groups of N = 2 and N = 3 .All of the subjects were further divided into four groups according to the number of disease vessels N = 0, 1, 2, 3). The serum cystatin C levels were 0.86 ± 0.17, 0.90 ± 0.16, 0.92 ± 0.17 and 0.97 ± 0.19 mg/L in the groups with disease vessels of 0, 1, 2, and 3, respectively, and the serum levels of cystatin C increased as the number of stenotic vessels increased (, 2, 3. TIn the present study, we demonstrated that in asymptomatic MetS patients without prior diagnosed CAD and with normal creatinine-based eGFR, elevated serum cystatin C was independently associated with the presence of asymptomatic CAD, even in an adjusted model. Serum cystatin C levels were positively and significantly correlated with the Gensini score. Furthermore, a positive association was also illustrated between serum cystatin C concentrations and the number of coronary arteries with luminal stenosis vessels. To the best of our knowledge, the study is the first to identify cystatin C as an independent risk factor for the development and severity of asymptomatic CAD in subjects with MetS and normal creatinine-based eGFR.It is well recognized that patients with impaired renal function are at significantly higher risk for cardiovascular disease, congestive heart failure, all-cause mortality and adverse long-term outcomes in contrast to patients without renal disease -27. CystAlthough cystatin C was independently associated with the presence of asymptomatic CAD,in the ROC analysis, the AUC for cystatin C falls into the range 0.6–0.7, which indicates the predictive value of cystatin as a sole marker was only moderate, and the cut-off value of 0.825 mg/L can not be categorized as ideal value to identify asymptomatic CAD effectively. Nevertheless, the addition of cystatin C may compensate for the inadequacy of. other classical parameters such as FRS for asymptomatic CAD. Future studies should investigate the feasibility of combined use of cystatin C and other parameters to improve the predictive value the presence of asymptomatic CAD.In addition, when the associations between cystatin C and severity of asymptomatic CAD were evaluated, we found a positive relationship between serum levels of cystatin C and the number of diseased vessels. A positive association was also found between serum cystatin C levels and the Gensini score independent of eGFR, even after adjustment for established risk factors associated with cardiovascular disease and cystatin C. Similar results were also reported by Niccoli et al. , who demDetailed mechanisms underlying link between cystatin C and asymptomatic CAD have not been fully elucidated. Renal mechanism seems to be a plausible link between increased cystatin C and asymptomatic CAD. Serum cystatin C was assumed to be a sensitive indicator of“pre-clinical” renal disease which can not be detected by eGFR based on serum creatinine . CystatiBesides renal mechanism, another possible explanation for the close relationship between cystatin C and. asymptomatic CAD is that cystatin C is associated with inflammation regardless of renal function Inflammation is a critical step in the development of atherosclerosis. Inflammatory cytokines associated with atherosclerosis may alter the relationship between cysteine protease and their endogenous inhibitors like cystatin C. The imbalance of cysteine protease and its inhibitor may increase the degradation of extracellular matrix and migration of monocytes, macrophages and vascular smooth muscle cells into the intima, thus leading to the development of atherosclerosis ,35. In tUric acid has been shown to be related with increased production of oxygen free radicals, to promote the oxygenation of LDL-c and to facilitate lipid peroxidation. StudiesThere were some limitations in our study which should be considered. First, we did not collect 24-hour urine for direct measurements of GFR because of its difficulty in daily practice. Instead, we assessed renal function with the commonly used Cockcroft-Gault formula which has some minor flaws that may lead to substantial bias in the assessment. However, numerous studies have demonstrated that the creatinine-based eGFR provides a good approximation. Second, to avoid confounding, we adjusted for well-known confounders such as gender, age, smoking status, and BMI. However, residual confounding may have occurred. Third, our results are derived from a cross-sectional study. Further studies, especially larger, population-based prospective studies are required to validate the findings suggested by the current study.In conclusion, our findings demonstrate that cystatin C was significantly associated with the presence and severity of asymptomatic CAD in MetS patients with normal kidney function. Probably cystatin C is more than a marker of glomerular filtration rate. Further research is warranted to clarify the pathophysiologic mechanisms responsible for this association.The authors declare that they have no competing interests.X Qing, W Xuping participated in the design of the study, W Furong, L Yunxia performed the statistical analysis, X Qing drafted the manuscript. Z Jian, X Qing contributed to the acquisition of data and its interpretation. G Ling, X Qing conceived of the study. All authors read and approved the manuscript.


A requirement for T286 autophosphorylation also for efficient Ca2+/CaM-stimulated CaMKII activity has been described, but remains controversial.Autophosphorylation of the Ca2+/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca2+/CaM-stimulated phosphorylation for two different peptide substrates (to ∼75–84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca2+/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.In order to determine the contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity is mildly (∼1.2–1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity.These results indicate that Ca This requires CaM binding not only to the subunit acting as kinase but also to the subunit acting as substrate (in order to make T286 accessible for phosphorylation) 2+/CaM-stimulated CaMKII activity. Beyond mere interest in the detailed regulation of an important signaling molecule, these findings support the interpretation that the phenotype of CaMKII T286A mutant mice (which show impaired LTP and learning) Here, the substrate phosphorylation rates of CaMKII wild type and its T286A mutant were compared directly in order to determine the relative contribution of T286 autophosphorylation to stimulated CaMKII activity. The results indicate a significant but very modest (∼15–25%) contribution of T286 phosphorylation to maximal CaCaM and CaMKIIα was purified after bacterial or baculovirus/Sf9 cell expression HEK 293 cells were transfected with GFP-CaMKIIα expression vectors by the calcium phosphate method , and harvested 72 h later. Cells were rinsed, scraped into ice-cold PBS, and collected by low speed centrifugation . Then, the cells were homogenized with a motorized pellet pestle (Kontes) for 10 sec in 0.4 ml ice cold 50 mM PIPES pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM DTT, and complete protease inhibitor (Roche). Debris was removed by centrifugation . A total of 12 CaMKII extracts were prepared in two separate rounds, with 3 extracts of each CaMKII wild type and T286A prepared in parallel in each individual round. CaMKII concentration was determined by their GFP-fluorescence measured in a spectrofluorometer  at 488 nm excitation and 510 nm emission wavelength (based on at least three samplings with increasing extract concentrations). This method allows a very accurate comparison of the relative CaMKII concentrations in the different extracts; absolute concentrations were derived by comparison to a GFP-CaMKII standard , 1 µM okadaic acid, and 75 µM substrate peptide . Under these conditions  were used where indicated (in Kinase activity assays (1 min at 30°C) were started by adding CaMKIIα (2.5 nM subunits) to a reaction mix containing 50 mM PIPES pH 7.2, 0.1% BSA, 1 µM CaM, 1 mM CaCl used in , the Ca2cated in . Reactio2+ with EGTA (and with subsequent reaction times and temperatures as indicated), or (ii) after reactions with naïve CaMKII essentially as in the kinase activity assays (with 1 µM CaM but with unlabeled ATP and without substrate peptide).CaMKII phosphorylation at T286 and T305 was assessed by Western-analysis with phospho-selective antibodies (PhosphoSolutions) as described 2+/CaM-stimulated for T-site binding “T-substrates” (such as AC2) 2+/CaM -stimulated phosphorylation of syntide 2 compared to AC2 as an underlying mechanism; for both CaMKII wild type and for its T286A mutant, Ca2+/CaM-stimulated syntide 2 phosphorylation occurred with an approximately 2fold higher Vmax compared to AC2 phosphorylation We have previously described the mechanisms underlying low CaMKII autonomy that is significantly further CaM-stimulated for regular “R-substrates” (such as syntide 2) compared to higher autonomy that is only mildly further CaIn order to distinguish if the different activity of the purified CaMKII wild type versus T286A mutant was due to the individual kinase preparations or was instead caused by the mutation, we directly compared the activity of the two different kinase forms in six different extracts for each kinase . Again, Next, we decided to compare the activity of CaMKII wild type and the T286A mutant also at submaximal stimulation (0.1 µM instead of 1 µM CaM) using syntide 2 as substrate. As expected, T286 autophosphorylation of CaMKII wild type was slower at such submaximal stimulation, but still reached the same level within the 1 min reaction time . Submaxi2+/CaM-stimulated conditions was not enhanced by submaximal Ca2+/CaM-stimulation conditions.In summary, the significant but mild (∼1.2–1.3fold) contribution of T286 autophosphorylation to stimulated activity under maximally Ca2+/CaM-binding region of the CaMKII regulatory domain  and with no appreciable phosphorylation on ice even after 30 min  2+/CaM-stimulation In order to explain the rational of our next experiments, several background issues need to be addressed first. The T305/306 residues are located within the Cay domain . T305/30r 30 min . Previou2+/CaM during 1 min stimulated activity assays prevents significant contribution of this inhibitory phosphorylation to the kinase activity measured 2+/CaM (which suppresses T305/306 phosphorylation), phospho-T286 may suppress T305/306 phosphorylation even further, as it slows down the Ca2+/CaM off-rate from ∼130 min−1 to <0.06 min−1, which should prevent any appreciable un-binding of CaM in the presence of Ca2+ (“CaM trapping”) 2+/CaM-stimulated activity. Contrary to this prediction, in a direct comparison to CaMKII wild type, the T286A mutant instead showed even less T305 autophosphorylation (less than one third compared to wild type) during 1 min reactions at 30°C . Under our assay conditions , no appreciable lag-phase is observed 2+/CaM-stimulated activity for a T286A mutant, but again only at the low 2 µM nucleotide concentration (using ATPγS) 2+/CaM-stimulated CaMKII activity in vitro translation system  2+/CaM-stimulated CaMKII activity was detected either The results of this study indicate that CaMKII autophosphorylation at T286 . Several partial crystal structures of CaMKII in its auto-inhibited basal state are now available 2+/CaM interaction CaMKII made “autonomous” by T286 phosphorylation is significantly (∼3–6fold) further stimulated by Ca


G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity. G protein-coupled receptors (GPCRs) represent the largest family of integral membrane proteins and regulate a wide variety of physiological processes. GPCRs typically bind to an extracellular agonist which causes the receptor to adopt an active conformation. However, some receptors exhibit constitutive activity in the absence of a ligand. The level of constitutive activity varies among receptors and also seems to depend on the cell type s-coupled GPCRs Xenopus oocytes GPR3, GPR6, and GPR12 constitute a family of constitutively active G1 dopamine receptor is an example of another GPCR that produces cAMP following internalization to support acute dopaminergic signaling s-dependent signaling, there is evidence that Gi-dependent signaling stimulated by the S1P receptor may occur internally s/Gi-coupled receptors or if it is a characteristic of only a few receptors and/or only occurs in certain cell types.An important mechanism that regulates GPCR signaling is desensitization. This process involves the G protein-coupled receptor kinases (GRKs) and the β-arrestins GPR3 behaves like agonist-occupied receptors in that it uses traditional GPCR pathways to transmit signals and is internalized by an endocytic pathway 2/95% air incubator. Cells plated in 6-well dishes were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) following the manufacturer’s recommendations. Experiments were performed 24 hr after transfection. Bisindolylmaleimide I (Bis I) and Phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem  and Tocris Bioscience , respectively. Methyl-β-cyclodextrin was purchased from Sigma .HEK293 cells were cultured in Dulbecco’s modified Eagle’s (DMEM)/F-12 medium  supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen) at 37°C in a humidified 5% COMouse GPR3-RFP was provided by Y. Saeki (Ohio State University) in pHGCX Human GPR3 in pCMV6-AC-HA-His (GPR3-HA) was purchased from Origene . Dynamin 1 wildtype (WT) and K44A in pcDNA3.1 were purchased from ATCC . GRK2 was provided by R. Lefkowitz  in pRK5. β-arrestin-2-GFP was obtained from M. Caron  in pS65T and was subcloned into pSP64.5. pcDNA3.1 empty vector was purchased from Invitrogen. CFP-Epac1(δDEP-CD)-YFP was provided by K. Jalink (The Netherlands Cancer Institute).Cells were washed in RPMI-1640 without bicarbonate (Invitrogen) +0.2% BSA (Sigma), pH 7.4, for 30 min at 37°C. Cells were incubated with 50 µg/mL Alexa Fluor 488-labelled transferrin (Invitrogen) for 2 min at 37°C, placed on ice for 10 min, and washed with cold PBS. Cells were fixed in 2% formaldehyde (Sigma) for 30 min at room temperature, washed twice with PBS, and transferrin internalization was examined with a Zeiss LSM 510 confocal microscope. Fluorescence was excited at 488 nm and detected at 530 nm. Images were collected using a 40X NA 1.2 water immersion objective .20) for 15 min each. Glutathione was quenched by 3 washes of iodoacetamide buffer  for 15 min each at 4°C.Biotinylation was performed on cells in suspension according to the manufacturer’s recommendation. Cells were plated in 6-well dishes and transfected as described previously. Cells were trypsinized using 0.025% Trypsin-EDTA, resuspended in growth medium, washed three times with ice cold PBS (pH 8.0), incubated in 0.5 mg/mL sulfo-NHS-LC-Biotin  for 30 min at 4°C, and washed with ice cold PBS (pH 8.0) +100 mM glycine to remove unbound biotin. For internalization assays, cell surface proteins were biotinylated with cleavable sulfo-NHS-SS-biotin (Pierce) as indicated above. Cells were then incubated in regular medium at 37°C for 30 min or 1 hr to allow internalization. Cleavage of surface biotin was performed as previously described Protein samples were prepared in RIPA buffer  containing protease inhibitor cocktail (Pierce Biotechnology), sonicated, and rotated at 4°C for 30 min. Supernatants were collected following centrifugation at 14,000 × g for 20 min at 4°C. Protein concentrations were determined by BCA assay (Pierce Biotechnology).Cell surface biotinylated proteins were precipitated from cell lysates using streptavidin agarose resin (Pierce). Sixty µg of protein was added to 30 µl of agarose beads in a total volume of 200 µl with RIPA buffer. Samples were rotated for 2 hr at 4°C, spun at 1150×g for 5 min at 4°C, and washed four times with 500 µl RIPA buffer. After the final wash, the beads were resuspended in Laemmli sample buffer with 5% 2-Mercaptoethanol and boiled for 5 min. Five µl of the sample was analyzed by Western Blot. 0.5 µg of total lysate was used to examine total GPR3 expression and GAPDH or β-actin as a loading control. The same sample was used to examine surface and total GPR3.Western blot analysis was performed as described previously HEK293 cells were transfected with the indicated plasmid DNA using Lipofectamine 2000. Twenty-four hr after transfection, cells were trypsinized as described above and 400,000 cells were washed with PBS, lysed in 225 µl of 0.1 M HCl for 10 min at room temperature, sonicated, and stored at −80°C until the assay was performed. Levels of intracellular cAMP were measured using the cAMP enzyme immunoassay (EIA) kit, Direct , using the non-acetylation EIA procedure. Similar results were obtained using both EIA kits. Cyclic-AMP was measured from ∼170,000 cells in 100 µl of 0.1 M HCl in all experiments unless otherwise noted. Results are presented as pmol/mL of cAMP.2, 1 mM MgCl2, and 20 mM HEPES, pH 7.4 at room temperature, and placed on a Zeiss Axio Observer A1 microscope equipped with Plan-Apochromat 63×/1.4 oil immersion objective, Polychrome V light source, DV2 DualView beam splitter and CoolSNAP-HQ2 CCD-camera . The YFP/CFP emission ratio upon 436 nm excitation  was measured before and after saturation of the sensor with isoproterenol plus isobutylmethyl-xanthine (IBMX) or forskolin plus IBMX. Measurements were monitored online and recorded by the VisiView software (Visitron). After each measurement, emission values were corrected for bleedthrough of CFP into YFP channel and for photobleaching as described in vitro50 * {(y-Rmin)/(Rmax-y)}1/n, where EC50 is the EC50-value of the sensor for cAMP determined in vitro min = 0, and Rmax = 100. The resulting equation [cAMP] = 13.21*y/(100-y) which describes the published curve, was used to calculate cAMP concentrations. Values are presented as mean ± SEM.To study changes in intracellular cAMP by FRET, HEK293 cells were plated on round glass cover slides and transfected 24 hr later with the CFP-Epac1(δDEP-CD)-YFP GPR3-HA mutants, in which serine and threonine residues in the third intracellular loop and C-terminus were mutated to alanine, were generated using the QuikChange II site-directed mutagenesis Kit  according to the manufacturer’s protocol. Amino acids at 237 and 242 in the third intracellular loop  and amino acids at 316, 317, 318, 324, 326, 328 in the C-terminus (1–6) were mutated to alanine. HPLC purified primers for mutagenesis were purchased from Sigma  and mutat test as described in each figure legend. Statistical analysis was performed using Graph Pad Prism software and significance was assessed at P<0.05.Data are representative of at least three independent experiments, unless otherwise specified. Values were analyzed by One-way ANOVA, Repeated Measures ANOVA, or Student’s To confirm that GPR3 is endocytosed in HEK293 cells, cell surface proteins were biotinylated with cleavable sulfo-NHS-SS-biotin and incubated at 37°C to allow for internalization. Following internalization, biotinylated proteins remaining at the cell surface were stripped and internalized biotinylated proteins were detected by streptavidin precipitation and Western blot. Biotinylated GPR3 was detected at the cell surface  Epac-based cAMP sensor(CFP-Epac1(δDEP-CD)-YFP In order to corroborate the cAMP data obtained by EIA assay, we performed aFRET-based assay using a low affinity-encoded receptor US28, which signals at the cell surface and is constitutively phosphorylated by GRKs. Following phosphorylation, US28 is rapidly internalized through a clathrin-mediated mechanism, which may not be dependent on β-arrestins GPR3 contains two potential sites for phosphorylation by protein kinase C (PKC) as well as several serine residues that could be targets for GRKs. To determine if GPR3 activity is regulated by any of these sites, we mutated all six serines in the C-terminus and the serine and threonine residues in the third intracellular loop to alanine . If GPR32-adrenergic receptor (β2AR) forms a complex with Gs and adenylate cyclase soon after leaving the ER in transit to the Golgi 2AR does not activate Gs at this compartment because it does not interact with its ligand until it reaches the cell surface. Since GPR3 is thought to signal in the absence of a ligand, it is possible that it could signal internally prior to membrane insertion; however, this has not been directly examined. It has been shown that inhibition of exocytosis in Xenopus oocytes results in premature meiotic resumption Together, these results demonstrate that the ST/A and S1-6A residues have different roles for cAMP signaling but both are important for surface expression of GPR3. It is not understood why the mutants exhibit lower surface expression. It is possible that the mutated receptors do not make it to the membrane as efficiently as the WT. Because decreased cell surface expression of the mutants does not prevent the receptor from producing cAMP, it suggests that either surface localization is not a requirement for cAMP production or that there is enough of the mutated receptor at the plasma membrane to produce cAMP at comparable levels to GPR3 WT. Although our previous findings support the hypothesis GPR3 does not signal following internalization, it is conceivable that some GPR3 may be able to signal internally prior to plasma membrane insertion. There is evidence that GPCRs preassemble with signaling components before reaching the cell surface. For example, the βOne possibility that may explain why the ST/A mutant is hyperactive is that it is resistant to desensitization. To determine if the serine and threonine residues in the third intracellular loop are required for silencing of GPR3 signaling by GRK2 and β-arrestin-2, cAMP levels were measured in cells co-transfected with the GPR3 ST/A mutant and GRK2 and β-arrestin-2. We found that cAMP levels produced by the GPR3 ST/A decreased in response to GRK2 and β-arrestin-2 overexpression. cAMP levels decreased by ∼76% and ∼73% for GPR3 WT and ST/A, respectively . ContrarBecause the ST/A mutant produced higher levels of cAMP but did not have increased cell surface expression, it raises the question of whether desensitization and internalization of GPR3 are distinct processes. To address this, we examined the individual effects of GRK2 and β-arrestin on cAMP production. We found that overexpression of GRK2 by itself is sufficient to significantly decrease cAMP production by GPR3 WT and GPR3 ST/A to the same extent as GRK2 with β-arrestin-2 . HoweverS activation and the steady-state levels of activated GS, thereby leading to prolonged generation of cAMP from endosomal compartments s activation and cAMP production. In the presence of GRK2, either this association may not occur, or the complex is internalized and desensitized. Interestingly, we did not detect an increase in cAMP when β-arrestin was overexpressed with the ST/A mutant, suggesting that an interaction between GPR3 and β-arrestin is required for cAMP production. Future studies are required to understand the role of β-arrestin in GPR3 signaling and trafficking and the importance of phosphorylation for these processes.Surprisingly, overexpression of β-arrestin alone significantly increased cAMP levels for GPR3 WT  but had The GPR3 ST/A mutant is susceptible to desensitization by GRK2 and β-arrestin overexpression; therefore, it is possible that other sites within GPR3 are phosphorylated by GRK or that GRK mediates desensitization independently of phosphorylation. To test this, we constructed a catalytically inactive GRK2 mutant (GRK2-K220R), in which the lysine at residue 220 was mutated to arginine. This mutation in the ATP binding domain has been shown previously to inhibit the kinase activity of GRK2 q-coupled receptors or by Gβγ and there is currently no evidence that GPR3 can signal through these pathways.As mentioned above, it is possible that other kinases are involved in regulating GPR3 activity. In addition to GRK, second messenger-dependent kinases including PKA and PKC can also phosphorylate GPCRs In summary, GPR3 is a constitutively active receptor that is an important regulator of meiosis in oocytes


Oral administration of sodium tungstate has shown hyperglycemia-reducing activity in several animal models of diabetes. We present new insights into the mechanism of action of tungstate.We studied protein expression and phosphorylation in the liver of STZ rats, a type I diabetes model, treated with sodium tungstate in the drinking water (2 mg/ml) and in primary cultured-hepatocytes, through Western blot and Real Time PCR analysis.Tungstate treatment reduces the expression of gluconeogenic enzymes  and also regulates transcription factors accountable for the control of hepatic metabolism . Moreover, ERK, p90rsk and GSK3, upstream kinases regulating the expression of c-jun and c-fos, are phosphorylated in response to tungstate. Interestingly, PKB/Akt phosphorylation is not altered by the treatment. Several of these observations were reproduced in isolated rat hepatocytes cultured in the absence of insulin, thereby indicating that those effects of tungstate are insulin-independent.Here we show that treatment with tungstate restores the phosphorylation state of various signaling proteins and changes the expression pattern of metabolic enzymes. Its main advantages are its low toxicity profile and the fact that it is active when administered orally. Oral administration of sodium tungstate to streptozotocin (STZ) -induced diabetic rats causes a dramatic decrease in blood glucose concentration Previous studies in our group demonstrated that tungstate increases glycogen deposition in primary cultured hepatocytes, thereby exerting insulin-like actions in these cells. Analysis of the effects of this compound on several components of the insulin signaling transduction cascade in cultured cells demonstrated that they are not mediated by the insulin receptor (IR) as its phosphorylation state remains unchanged after treatment in vivo on several pathways potentially implicated in the improvement of the diabetic state. For this purpose, in STZ-induced diabetic rats treated with tungstate, we analyzed changes in the phosphorylation and the expression of key enzymes and transcription factors involved in the regulation of gluconeogenesis, one of the most relevant processes altered in the liver of diabetic patients. Here we show that a 30-day treatment with this compound restores the phosphorylation state of various signaling proteins and changes the expression pattern of regulatory enzymes.Here we studied the mechanism of action of tungstate All procedures were approved by the Barcelona Science Park's Animal Experimentation Committee  and were carried out in accordance with the European Community Council Directive and the National Institute of Health guidelines for the care and use of laboratory animals.Sodium tungstate was from Carlo Erba . STZ was from Sigma-Aldrich . Enzymes and biochemical reagents were from either HORIBA ABX or Sigma-Aldrich. All other chemicals were of analytical grade. ERK antibody was from Upstate . GSK3α/β antibody was from Santa Cruz Biotechnology . PKB/Akt antibody was from Sigma-Aldrich. Phospho-GSK3α/β (Ser 21/9), phospho-ERK (Thr 202/Tyr 204), phospho-p90rsk (Ser 380) and p90rsk antibodies were from Cell Signalling . Phospho-PKB/Akt (Ser 473) antibody was from NanoTools . GAPDH antibody was from Ambion . PEPCK antibody was a kind gift of Dr. Daryl Granner .ad libitum. When stated, diabetes was induced by a single intravenous injection of STZ (60 mg/kg of body weight) in 0.9% NaCl with 100 mM sodium citrate buffer (pH 4.5). Diabetes was confirmed by determination of glycemia . Diabetic rats were used 5–7 days after STZ injection. At the beginning of the experiment, the animals were divided into four groups: control (Ctrol), diabetic (Diab), control treated with tungstate (Ctrol+W) and diabetic treated with tungstate (Diab+W). The non-treated animals were given normal drinking water while the treated ones received a solution of 2 mg/ml sodium tungstate in distilled water. The treatment was carried out for 30 days. During this period, glycemia, fluid and food intake were measured every three days between 10 and 12 a.m. At the end of the experiment, rats were anesthetized with Isofluorane 2%  and heart blood was collected to measure serum parameters. Afterwards, organs were rapidly frozen in liquid nitrogen for later determinations. Animals were sacrificed between 10 and 12 a.m.Adult male Wistar rats (200 g) were kept under a constant 12-h light-dark cycle and were allowed to eat and drink GS activity was determined in frozen liver samples homogenized (Polytron) in 10 volumes of ice-cold 10 mM Tris-HCl buffer (pH 7.4) containing 150 mM KF, 15 mM EDTA, 0.6 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 25 nM okadaic acid and 15 mM 2-mercaptoethanol. Whole homogenates were used for determinations. Total (T) GS activity (measured in the presence of 6.6 mM G6P), intrinsic (I) GS activity (measured in the absence of G6P) and GS I/T activity ratio (−G6P/+G6P) were measured as described Homogenates (20 µg of protein) were resolved by 10% SDS-PAGE. The protein was transferred onto a nitrocellulose membrane and probed with the mentioned primary antibodies. Secondary antibodies conjugated to horseradish peroxidase against rabbit , mouse (DakoCytomation) or sheep (DakoCytomation) immunoglobulins were used. Immunoreactive bands were visualized using an ECLplus kit , following the manufacturer's instructions. Densitometric analyses were carried out using NIH Image J software.Blood glucose was measured using an Ascensia Brio Glucose Analyzer (Bayer). Serum glucose (HORIBA ABX) and triglyceride (Sigma-Aldrich) concentrations were measured spectrophotometrically by standard techniques adapted to a COBAS Mira analyzer (Roche Diagnostics). Serum insulin levels were measured by immunoassay (Spi Bio).Collagenase perfusion was used to isolate hepatocytes from male Wistar rats (180–225 g) previously fasted for 24 h, as described 6 cells per plate) following the hepatocyte isolation and culture method described in the previous paragraph. After incubating the cells overnight in serum- and glucose-free DMEM, the medium was replaced with 3 ml of glucose production buffer consisting of glucose-free DMEM supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate Primary hepatocytes were cultured in p60 plates , in the presence of 50 ng of random hexamers.Total RNA was isolated from rat liver tissue by homogenizing (Polytron) 100 mg of sample in 1 ml of TRIzol (Invitrogen). After centrifugation at 12,000×CtΔΔ method using 18S rRNA as endogenous reference.PCR tests were performed at the Genomic Unit of Core Scientific Services at the University of Barcelona following the standard Real-time PCR protocol of the ABI Prism 7900 Detection System, together with the appropriate ready-made TaqMan primer/probe sets (Applied Biosystems). Each sample was analyzed from triplicate wells. The temperature profile consisted of 40 cycles of 15 s at 95°C and 1 min at 60°C. Data were analyzed with the 2t test using Microsoft Excel . Statistical significance was assumed at P≤0.05.Data are expressed as the mean ± S.E.M. Statistical significance was determined by unpaired Student's Healthy and STZ-diabetic rats were treated for 30 days with oral sodium tungstate (2 mg/ml) dissolved in distilled water. Non-treated animals of each group were given tap water. We observed a slight decrease in water intake in healthy treated animals compared to the non-treated ones. In the case of diabetic treated animals, there was a drastic decrease in water intake compared to their controls . As prevA key effect of the 15-day treatment with tungstate in the STZ rat is a normalization of hepatic phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels One of the hallmarks of tungstate is its capacity to increase ERK phosphorylation in several cell lines Using Western blot analysis with phospho-specific antibodies, we first observed that ERK phosphorylation is dramatically decreased in livers of STZ diabetic rats, at two (data not shown) and five weeks after STZ injection . HoweverWe also analyzed the phosphorylation state of several proteins of the insulin signaling cascade. In addition to ERK phosphorylation, p90rsk and GSK3 phosphorylation was also decreased in the livers of STZ-diabetic animals . TungstaIn order to identify the mechanism of action by which tungstate decreases the expression of gluconeogenic enzymes, we analyzed whether this compound also modifies the expression of the main transcription factors and coactivators involved in the regulation of PEPCK and G6Pase, the rate-limiting enzymes of gluconeogenesis. The phosphorylation of ERK-1/2 and of GSK3 induced by tungstate in livers of STZ rats led us to focus first on the expression of c-jun and c-fos, which act downstream of ERK and GSK3 and are involved in the negative regulation of hepatic gluconeogenesis Next, we analyzed the expression of two other well-known transcriptional regulators of gluconeogenic enzymes, forkhead box 1 (FoxO-1) and hepatic nuclear factor-4 alpha (HNF-4α) Tungstate treatment ameliorated hyperglycemia and improved the diabetic state by affecting hepatic metabolism and extra-hepatic targets. To differentiate the former from the latter, we performed experiments on primary cultured hepatocytes. In order to stimulate the gluconeogenic pathway, we treated rat primary cultured hepatocytes with tungstate in the absence or presence of dexamethasone The liver is a key organ in the regulation of metabolism, especially glucose homeostasis, in which hepatic gluconeogenesis is crucial. The expression of PEPCK in liver is greatly increased in the diabetic state, thus leading to enhanced glucose production and contributing to hyperglycemia. It has been reported that tungstate increases hepatic glycolysis and glycogen synthesis while reducing gluconeogenesis Several groups have described differences in the phosphorylation of ERK-1/2 in most tissues of STZ-induced diabetic rats. However, data are controversial, as increases and decreases have been reported in ERK phosphorylation in STZ models depending on tissue and time. For instance, ERK-1/2 phosphorylation is increased in the kidneys in vitro activation of glycogen deposition in response to tungstate is due to GSK3 inhibition mediated by ERK phosphorylation in vivoin vivo by the same mechanism.It has been proven that increased ERK activity leads to augmented p90rsk phosphorylation and consequently to that of GSK3 β inactivation leads to enhanced cellular c-jun levels Furthermore, ERK-1/2 activate c-fos through its phosphorylation as well as by increasing its expression in vivo are not a result of altered water intake. We observed moderately increased insulinemia in tungstate-treated STZ-diabetic rats, possibly as a result of the reported tungstate-mediated regeneration of pancreatic beta-cell population Tungstate treatment reduced water intake in both diabetic and healthy rats. The drastic reduction in water intake of treated diabetic animals can be attributed to the normalization of glycemia, since it occurred in parallel to the decrease in blood glucose levels. However, treatment of healthy animals with tungstate had no effect on the parameters measured, indicating that the effects observed in vitro model of induced gluconeogenesis, and may contribute to ameliorating hyperglycemia. By studying the mechanism of action of tungstate, we have pinpointed key therapeutic targets that control glucose homeostasis. These targets deserve further exploration in the search for more efficient treatments for diabetes.Taken together, our findings indicate that the antidiabetic agent sodium tungstate restores intrahepatic ERK phosphorylation, and the downstream signaling and expression of transcription factors, which in turn may regulate the expression of several metabolic genes in a rat model of diabetes mellitus. Our results indicate that tungstate modulates hepatic glucose production, one of the most relevant processes in the regulation of glucose homeostasis and one that is altered in diabetes. These actions appear to be independent of insulin signaling, as shown in an Figure S1Water intake for each experimental group during the 30-day treatment. Treated animals were given tungstate dissolved in distilled water, whereas non-treated animals were given tap water. Only diabetic and diabetic-treated animals were caged individually. Water intake was measured every 3 days for each cage. Measurements are expressed as the mean of ml/day per animal. Error bars represent S.E.M.(TIF)Click here for additional data file.


A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists.  •MS/MS identified >37,000 phosphorylation sites in adipocytes•Insulin regulates the phosphoproteome over a wide temporal timescale•Akt phosphorylates SIN1 on T86 in response to insulin•SIN1 phosphorylation activates a positive feedback loop between Akt and mTORC2 Insulin plays a major role in controlling metabolic homeostasis, cellular growth, proliferation, and survival, and defects in the mechanisms underlying these processes contribute to a range of diseases including type 2 diabetes, cardiovascular disease, and cancer. Protein phosphorylation plays a major role in almost all insulin-regulated processes. As a member of the receptor tyrosine kinase (RTK) family, the insulin receptor engages the canonical phosphoinositide-3 kinase (PI3K)/Akt  and mamm−/− mouse embryonic fibroblasts (MEFs) was assessed to a depth of 4,251 and 7,255 phosphosites, respectively (The mTOR-regulated phosphoproteome was recently investigated using quantitative mass spectrometry (MS). By examining the sensitivity to the mTOR inhibitors Torin1, rapamycin, and Ku-0063794, the mTOR network in HEK293 cells and Tsc2ectively . Surprisectively  or chemiectively  while ovTo characterize the insulin signaling network we applied two distinct quantitative phosphoproteomics strategies using stable isotope labeling with amino acids in cell culture (SILAC) . In the Our phosphopeptide enrichment was highly efficient and selective, since phosphopeptides consisted of an average of 83% of the total peptides identified from the phosphopeptide-enriched samples E and repIn parallel with the phosphoproteome studies, we retained a fraction of the peptides for total proteome analysis by MS following strong anion exchange chromatography C Wiśnie, allowinComparison of the proteome and phosphoproteome revealed an overrepresentation of phosphoproteins associated with the cytoskeleton, plasma membrane (PM), and nucleus, consistent with the importance of phosphorylation as a key functional modulator at these locations B. Enrich−8, Wilcoxon rank-sum test). Of course, phosphorylation is not limited to single sites within a protein, so even with near-exhaustive coverage of phosphorylated proteins, there will likely be many phosphorylation sites that remain out of reach with the current technology or that are only present at measurable stoichiometry following unique cellular stimulus.To further assess the phosphoproteome coverage of our data, we mapped the proteins found to contain phosphorylation sites onto the ranked proteome abundance A. We obsWe next compared our data set with a study performed in nine mouse tissues . We obseWe identified several phosphorylation sites known to be important to the actions of insulin and growth factor signaling in general, such as the activating T loop phosphorylation site of Akt (T308) and the hydrophobic motif (HM) of Akt1 (S473) not identified in the mouse tissue phosphoproteome study. The detection of these and other phosphorylation sites in our data highlights the importance of system perturbations using growth factors or other stimuli to activate signaling networks when studying the phosphoproteome. Under resting or starved conditions, the stoichiometry of many growth factor-regulated phosphorylation sites is known to be low, making their detection unlikely if not impossible.In the studies using PI3K and Akt inhibitors, we quantified 19,507 unique phosphorylation sites on 4,065 proteins  revealed 3,152 positively regulated and 1,574 negatively insulin-regulated phosphorylation sites on 1,285 and 830 proteins, respectively    of the tc-means) . To invec-means) . Examinac-means) .Interestingly, all well-characterized Akt substrates were found in the fastest clusters (A and B), together with the activation sites of Akt (T308 and S473), while p70S6K and its substrate (S6) were found together in slower clusters (clusters D–F) . This suA major challenge in studying protein phosphorylation is defining physiological kinase-substrate relationships. This is difficult in light of the loss of sequence specificity that occurs for many kinases in vitro. For example, the evolutionarily related AGC kinases Akt and p70S6K phosphorylate substrates with indistinguishable motifs in vitro , yet ourKinase-substrate prediction classifiers were trained for Akt, mTORC1, and PKA, since these kinases collectively represent a wide range of the actions of insulin and are implicated in a diverse range of biological processes of broad significance. Features used for classification were selected to describe three diverse characteristics of each substrate: (1) the amino acid sequence surrounding the phosphorylated residue, (2) the response of the phosphosite to the PI3K/mTOR and Akt inhibitors , and (3) the temporal profile of phosphorylation in response to insulin  and S4B.While the high-ranking Akt, mTORC1, and PKA predictions from our machine learning approach included many known substrates for each kinase, there were also many phosphorylation sites not previously recognized as part of the insulin signaling network. Many of these previously unrealized phosphorylation sites likely represent previously unrealized actions of insulin. For instance, putative mTORC1 sites in Ulk1 and Ulk2, the ubiquitin-specific processing protease 32 (Usp32), the La ribonucleoprotein domain family member 4B (Larp4B), the poly(rC)-binding protein 1 (Pcbp1), and topoisomerase II homolog 1 (Patl1)  suggest Candidate Akt substrates include butyrate response factor 2 (Brf2), an mRNA-binding protein that promotes mRNA deadenylation and degradation. A homologous site on Brf1 is a known Akt substrate , supportOne predicted Akt substrate of particular interest was SIN1, an indispensible subunit of mTORC2 D require−/− MEFs and selected for cells expressing SIN1 similar to endogenous levels. We found that SIN1 T86A was equally capable of forming mTORC2, since immunoprecipitation of Rictor copurified both the phosphomutant and WT SIN1 equally, suggesting that formation and stability of the complex was not impaired  by mTORC2. Consistent with this, Akt T450 phosphorylation was abolished in SIN1/− cells   Akt as the substrate. mTORC2 complexes obtained from cells pretreated with the Akt inhibitor MK2206, but not rapamycin, had impaired mTORC2 activity and a concomitant block of SIN1 T86 phosphorylation F. In thi−/− MEFs rescued with SIN1 WT, T86A, and T86E mutants and performing in vitro kinase assays. mTORC2 isolated from cells containing SIN1 T86A displayed an impaired growth factor increase in kinase activity  Akt translocates to the PM where its activity is increased by phosphorylation at T308 by PDK1; (3) mTORC2, probably via the pleckstrin homology (PH) domain in SIN1 (We propose the following model for the activation of mTORC2 H: (1) gr in SIN1 , also trHere we provide a quantitative atlas of dynamic protein phosphorylation and show that these large-scale phosphoproteomics data can be integrated using in silico approaches to delineate key topological features of this essential signaling network. These data serve as a resource for future hypothesis-driven research into both known and currently poorly defined actions of insulin and other growth factors that converge upon signaling nodes like Akt that are central to insulin action.While dysregulated signaling networks downstream of RTKs are a key feature of diabetes, cardiovascular disease, and many types of cancer, there is a paucity of systems-wide quantitative data delineating signaling networks in healthy cells. Hence, the data described herein brings us a step closer to a more complete understanding of disease phenotypes. It is apparent, for example, that the scale of growth factor-regulated protein phosphorylation, in terms of both functional and temporal distribution, is much greater than previously appreciated. This underscores the importance of classifying phosphorylation data using context-specific systems. Therefore, the current study, which focuses on insulin signaling in the adipocyte, will provide an ideal framework with which to move toward more complex signaling networks in tissues comprising multiple different cell types and multiple intercellular communication networks.Our data highlight the temporal dimension in protein phosphorylation as a key indicator of functional behavior and its modification by extrinsic cues such as growth factors, including insulin. Two major nodes regulated by insulin are mTOR and Akt, each of which controls distinct biological endpoints such as nutrient sensing, protein synthesis, metabolism, and cell survival. In our quantitative phosphoproteome, we observed substantial temporal resolution in the activation of these two pathways. The activation of Akt involves at least eight molecular events consisting of changes in protein localization, protein-protein interaction, and conformation. Despite this, Akt was fully active within 30 s of insulin stimulation, while the activation of mTORC1 was markedly delayed. The resolution of Akt and mTORC1 activity B and 4C To illustrate the utility of these data for the identification of kinase-substrate relationships, we employed a machine learning approach utilizing SVMs. We demonstrated that one of the predicted substrates, SIN1, is a physiological target of Akt. Moreover, we found that the site-specific phosphorylation of SIN1 T86 is responsible for the direct activation of mTORC2 that occurs in response to growth factors. This is intriguing since mTORC2 is typically considered to be upstream of Akt, as it phosphorylates Akt at the hydrophobic motif , therebyHere we have highlighted only a subset of the biological processes that are encapsulated in our data. A cursory analysis reveals numerous other important pathways that are likely to be regulated by insulin, as well as other RTKs that invoke the PI3K/Akt-mTOR pathways. Hence, we expect that many additional cellular targets of insulin will emanate from studies of this kind.2 for phosphopeptide analysis (18 stop and go extraction (STAGE) tips.3T3-L1 fibroblasts were triple SILAC labeled , differeanalysis . Eluted 18 ReproSil particles (Dr. Maisch GmbH). An EASY-nLC system was connected to the mass spectrometer with a nanospray ion source, and peptides were separated with a binary buffer system of 0.5% acetic acid (buffer A) and 80% MeCN plus 0.5% acetic acid (buffer B), at a flow rate of 250 nL/min. Peptides were analyzed on an Orbitrap Velos or Q Exactive benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific). Up to 10 peptides on the Orbitrap Velos or 15 on the Q Exactive were fragmented in the HCD cell and analyzed with high resolution  in the Orbitrap detector. Dynamic exclusion and lock-mass were enabled (m/z 445.120025).Eluted peptides were resuspended in MS loading buffer  and loaded onto a 20 cm column with a 75 μM inner diameter, packed in house with 3 μM CRaw mass spectrometry data were processed using the MaxQuant software  version Data analyses were performed using Microsoft Office Excel, the R software environment, and the bioinformatics platform Perseus . Gene ontology (GO) annotation enrichment analysis was performed in Perseus, and significance was assessed using Fisher’s exact test. The whole quantified phosphoprotein data set was used as a background data set, and the Benjamini-Hochberg FDR method was used for multiple hypotheses testing (FDR < 0.02). Motif analysis was performed using iceLogo , using pThe workflow of the analysis is shown in


St. Helens, Washington. The remaining δ34S values were similar to the isotopic composition of coal from southern Wyoming. The δ15N values in ice-core samples representing a similar period of snow deposition were negative, ranging from -5.9 to -3.2 ‰ and all fall within the δ15N values expected from vehicle emissions. Ice-core nitrate and sulfate deposition data reflect the sharply increasing U.S. emissions data from 1950 to the mid-1970s.Ice-core samples from Upper Fremont Glacier (UFG), Wyoming, were used as proxy records for the chemical composition of atmospheric deposition. Results of analysis of the ice-core samples for stable isotopes of nitrogen (δ High r of SO2 .x and SO2. Trends in precipitation chemistry at NADP/NTN sites in the western United States have indicated an increase in total N deposition and a decrease in 34S, were monitored in bulk snowpack samples collected from a network of 52 high-elevation sites in the Rocky Mountains from 1993 to 1999 [34S values indicated that snowpack S in high-elevation areas is primarily derived from anthropogenic sources [Thin soils and dilute surface-water systems in high-elevation areas have limited capacities to buffer increased acidity associated with the airborne contaminants of NO981-1998 . In addi34S values in firn and ice-core samples have been used to reconstruct changes in sulfate sources to central Asia, Greenland, and Antarctica. The variation in δ34S values in a firn core from central Asia allowed for the identification of S derived from marine evaporites (+15 ‰) during high dust deposition events and anthropogenic emissions (+5.4 ‰) [34S signatures in Greenland ice cores were comprised of marine biogenic emissions, continental dust sources, background volcanism, and continental biota [34S signatures in the ice-core samples indicated anthropogenic S sources.Changes in δ(+5.4 ‰) . Preindual biota . Beginni34S values that were used to confirm and contrast the different S isotopic signals between the low-latitude Agunge volcanic eruption in 1963 and background marine biogenic sulfate [34S values over the past 1,100 years, indicative of no temporal change in influencing sources of S [34S in two ice cores from east Antarctica over the complete glacial/interglacial cycle were significantly lighter than previous measurements of δ34S from the South Pole [34S values in an ice core collected from west Antarctica were found to be a mixture of marine and volcanic S sources during the time period from 1935-1976. [In Antarctica, shallow firn cores collected from the South Pole contained δ sulfate . Composi35-1976. .2O in trapped gases (ice cores) from Greenland and interstitial air (snowpack) from the South Pole have been used to differentiate between natural and anthropogenic sources [15N value of atmospheric N2O has dropped by 1.7 ‰ during the 20th century, likely due to increasing agricultural activities [2O(g) have indicated a -1.8 ‰ shift in δ15N over the last two centuries, primarily due to anthropogenic influences [15N of 15N  .15N  NADP/NTN data (sites WY06 and WY98) trends in the chemistry of atmospheric deposition with similar and longer term data preserved in glacial ice from the nearby UFG  were evaporated to dryness within covered canisters flushed by dry nitrogen. The samples were transferred from 4-L Pyrex beakers to smaller Savillex beakers when they had been reduced to about 100 mL. Drydown salts were redissolved in a few mL of deionized water which was pipetted into tin capsules and evaporated a few hundred microliters at a time. One to 2 mg of powdered V2 for analysis by isotope ratio monitoring using a Micromass Optima [34S values for them of -34.05 and 21.1 ‰, respectively [2+-Na+-K+ solutions were prepared with reagents of known isotopic composition and then dried and analyzed. Sodium-dominated solutions gave δ34S values indistinguishable from the reagents used to prepare them; however, solutions with Na+:Ca2+ ratios equivalent to the UFG samples were about 1 ‰ low. The cause of this inaccuracy is uncertain. No attempt was made to correct the UFG measurements for a possible similar inaccuracy. The S isotopic compositions are reported in δ-notation relative to Vienna Canon Diablo troilite. Reproducibility for triplicate analysis of sample ISO-1 was ±0.4 ‰ (one standard deviation).Filled capsules were combusted in an elemental analyzer to produce SOs Optima . The eleectively . To eval2O) was produced by the denitrifier method using the Pseudomonas chlororaphis bacterium (ATCC 43928) [3 (δ15N = 180 ‰), USGS34 KNO3 (δ15N = -1.8 ‰), and USGS35 NaNO3 (δ15N = 2.7 ‰) were analyzed along with the unknowns.For N isotopic analysis, meltwater volumes corresponding to 1.2 micrograms of nitrate (6-80 mL) were evaporated to <5 mL by the same method used for sulfur. Nitrous oxide (NC 43928) ,31, and 17O  [17O leads to δ15N results that are erroneously high by 1 ‰ per 18.8 ‰ increase in Δ17O [15N determinations for USGS35, which has a Δ17O value of 21.1 ‰, showed an error corresponding to 1 ‰ per 18.3 ‰ increase in Δ17O. The highest Δ17O values that have been observed to date in modern and ancient atmospheric nitrate are 39 and 20 ‰, respectively [17O of atmospheric nitrate is unlikely to exceed about 30 ‰ in the western United States [17O values of 30 ‰ or lower, the error in our δ15N results is no more than 1.6 ‰. The N isotopic compositions are reported in δ-notation relative to atmospheric N2(g). Reproducibility for triplicate analysis of USGS34 was ±0.27‰ (one standard deviation).Because the an zero) . For sin in Δ17O . Our δ15ectively -36, and d States . If the After filtration with 0.45 μm disposable polysulfone filters, a modified ion chromatography procedure for low ionic strength samples was used for the analysis of  and . A DioneAll chemical data from the ice-core analyses can be found in additional file Tritium, chloride-36, and carbon-14 age-dating methods ,39 were where D is depth below glacier surface, in m. This age-depth profile is in excellent agreement with known time horizons in the ice core that include (1) 1963 tritium and 1958 chloride-36 peaks from above-ground nuclear testing; 2) 1883 Krakatau volcanic eruption; (3) 1815 Tambora volcanic eruption; and (4) 1729 (± 95 years) carbon-14 age date of an insect leg entrapped in the ice  with a mean δ34S value of volcanic ash from the 1980 eruption of Mt. St. Helens (8.3 ‰) [- [Seven composite ice-core samples and one snow sample collected from UFG were analyzed for δ (8.3 ‰)  indicate (8.3 ‰)  and elev and Cl- . Ice sam and Cl- .34S value in the layer corresponding to atmospheric deposition from Mt. St. Helens, the remaining δ34S values in the ice-core samples ranged from 5.1 to 6.8 ‰   and mass(-2.4 ‰) . The δ3434S values that range from +9 to +33 ‰ [34S values found in UFG ice-core samples  may reflect contributions from lake-bed sulfate.The WRR and UFG are in close proximity to large expanses of arid and semi-arid lands composed of soils, sedimentary rocks, and dry lakebeds containing gypsum and anhydrite . Therefoto +33 ‰  and are e +7.5 ‰ , slightl34S composition of 5.1 ‰ for organic S [34S values in ice-core samples from UFG  and anthropogenic sources  [15N value in the UFG ice-core samples  was equal to the mean δ15N value of six archived precipitation samples collected during 2000 at NADP/NTN site WY98 . The similarity in mean δ15N values between the ice-core and NADP samples suggests similar Seven composite ice-core samples representing the time period of deposition from 1951 to 1988 were collected from UFG and analyzed for δ  figure . The δ15exhaust) . The mea15N values were compared with δ15N values from potential NOX sources from previously published data  and industrial combustion (12%). Data compiled for the western United States indicate that on- and off-road mobile sources comprise 67% of the anthropogenic NOx emissions, with stationary sources comprising only 22% of the anthropogenic NOx emissions [15N values with existing end-member data, it appears likely that vehicular NOX emissions may have a large impact on Ice-core δa figure . Although Africa , simulath Africa , or keroh Africa . Ice-corO values . The δ15missions ,50. Basen Agency , on- andmissions . Based ox emissions from stationary sources were strongly correlated with spatial variation of δ15N  to calculate deposition masses in kilograms/hectare (kg/ha). The age of each ice-core sample from site DH-98-4 was determined from the modified age-depth profile developed for the 1991 ice core to account for the additional snow deposited between 1991 and 1998. Once an age was assigned to each ice-core sample, the In addition to the S and N isotope data, the ice-core samples were used to provide an archive of annual and WY98 . Annual Comparison of the 0 figure . During x produced from the oxidation of nitrous oxide in the stratosphere should contain a δ15N of ~ 19 ‰ [15N  indicates an increasing trend in X and SO2 from 1950 to 1998  would provide additional insight into the existence of isotopic trends. Unfortunately, the low concentration of 34S values in seven ice-core samples representing snowfall deposited from approximately 1946 to 1988 were similar, ranging from 5.1 to 8.1 ‰. The highest δ34S value in the UFG ice-core samples was associated with snow deposited primarily during 1980 and coincident with the eruption of Mt. St. Helens, Washington, during May 1980. The measured δ34S value of 8.1 ‰ in the ice core was in close agreement with the mean δ34S value of 8.3 ‰ of volcanic ash from the 1980 eruption. The remaining δ34S values from ice-core samples were substantially enriched relative to biogenic 34S values of water-soluble 34S isotopic composition suggesting that power plant emissions may be a dominant source of S deposition to the WRR.Glaciers in the WRR present a unique opportunity to couple short-term (1980 to present) NADP/NTN data trends in the chemistry of atmospheric deposition with longer-term data, including stable isotope data, preserved in glacial ice. The δ15N value in the UFG ice-core samples  was equal to the mean δ15N value of six archived precipitation samples collected during 2000 at NADP/NTN site WY98, suggesting similar 15N values were substantially depleted relative to δ15N values of NOX from coal-fired powerplants, North American kerogen deposits, or simulated lightning and were substantially enriched relative to soil-released N. The ice-core samples were similar in isotopic composition to δ15N values measured from vehicle emissions. This similarity in isotopic composition is consistent with NOX emission data compiled for the U.S. during 1998 that indicates vehicle emissions as the dominant source of NOX.The mean δComparison of The authors declare that they have no competing interests.DN conceived the study, designed and coordinated study execution, assisted with sample processing, and drafted the manuscript. PS assisted in the design and execution of the study, assisted with sample processing, carried out the anion analyses, and assisted with the interpretation of the data. CJ carried out the isotopic analyses and provided assistance in drafting the manuscript. All authors read and approved the final manuscript.Supporting data. Isotopic and anion data used in the studyClick here for file


In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals  CXCL12 regulates important hematopoietic functions, induces cell adhesion and chemotaxis, and coordinates the circulation of hematopoietic stem cells, lymphocytes and monocytes CXCL12 elicits cellular responses by binding to CXCR4 and CXCR7, which are seven transmembrane receptors (7TMR) that activate G-proteins By binding to its receptors, CXCL12 induces another interesting phenomenon, i.e. the transactivation of the human EGF receptor 1 (HER1) and other HER family members. Such a crosstalk is a general function of 7TMR signaling in vitro model for studying both HER1 and CXCR4 signaling In HERs/CXCR4 double-positive cells, a transactivation of HERs along intracytoplasmic pathways is possible and was described The 5637 (bladder carcinoma) and HeLa  human cell lines, purchased from American Type Culture Collection, were grown in DMEM+10% FCS  until confluence and were subsequently detached by accutase .Cells under basal conditions or following stimulation for 20 minutes with 200 ng/mL CXCL12  or 25 ng/mL HB-EGF  were detached and incubated with anti-human PE-tagged CXCR4 or HER1 mAbs, as well as appropriate isotype controls . Cells were analyzed on a FACSCalibur  flow cytometer running FlowJo 8.8.2 software .Semiconfluent, 24-hour starved cells were incubated in the presence or absence of 25 ng/mL recombinant human HB-EGF or 200 ng/mL of either recombinant human CXCL12 or chemically synthesized [N33A]CXCL12 . In selected experiments, HeLa cells were prestimulated for 1 minute with either 200 ng/mL recombinant human CXCL12 (BD PharMingen) or chemically synthesized [N33A]CXCL12 and subsequently stimulated with 25 ng/mL HB-EGF. Cell pellets were obtained to perform protein extractions at different treatment times ranging from 1 to 15 minutes.Cell pellets were lysed for 30 minutes in 1 mL of ice-cold cell extraction buffer  that was supplemented with a protease inhibitor cocktail  and 1 mM PMSF (Sigma). After centrifugation at 13,000 rpm for 10 minutes at 4°C, aliquots of supernatants were stored at −80°C until used.Total HER1 and HER1 pY1068 or pY1173, and total ERK1/2 and ERK1/2 pTY185/187 were evaluated in cell protein extracts by using commercially available ELISA kits (Biosource). The results were expressed as percentages of phosphorylation at the specified times after normalization as phosphorylated molecule/total molecule ratios.7 cells, either unstimulated or stimulated with 25 ng/mL HB-EGF for 15 minutes, were harvested and underwent HER1 immunoprecipitation, reduction, alkylation and tryptic digestion. Ten microliter aliquots of each resulting peptide mixture were analyzed by LC-MS/MS (liquid chromatography coupled to an ion trap mass spectrometer) system Around 1×10vs. unstimulated cells was measured by MTT  incorporation, as previously described Approximately 2,500 cells/well were seeded in 96-well plates and allowed to adhere for 24 hours, prior to incubation with 25 ng/mL HB-EGF and/or 200 ng/mL recombinant human CXCL12 and/or 1 µg/mL cyclosporin A . After 72 hours, the proliferation of stimulated 5) and transfected 72 hours using HiPerFect Transfection Reagent and siRNA  as previously described To silence the expression of β-arrestin 2, 5637 or HeLa cells were seeded on 12-well plates propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl)maleimide methanesulfona-te salt  or 44 µM N-(4-Aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride ,) or 30 µM 1--4-phenylpiperazine  or 1 µg/mL Cyclosporin A ,) – specific inhibitors of PKC, calmodulin, CaMKII and calcineurin, respectively. HeLa cells were then treated with 200 ng/mL human recombinant CXCL12 (BD PharMingen) or chemically synthesized [N33A]CXCL12 (BD PharMingen) for up to 15 minutes or for 1 minute before stimulation with 25 ng/mL HB-EGF for up to 15 minutes.Student's t-test, Mann-Whitney U test and Kruskall-Wallis ANOVA by ranks were used. Differences were considered significant for p values <0.05.via binding of a shedded ligand would ultimately be indicated by HER1 internalization, we concluded that CXCL12 did not induce HER1 transactivation in 5637 or HeLa cells through this mechanism. Another approach implies evaluating the phosphorylation of HER1 and downstream of HER1 associated with different stimuli.Like other epithelial cells, 5637 and HeLa cells co-express HER1 and CXCR4 under basal conditions . For stiTo demonstrate the full sensitivity of 5637 and HeLa cells to HER1 stimulation, we assessed the full pattern of HB-EGF-induced HER1 phosphorylation at tyrosine  and serine (S1142) residues by mass spectrometry analysis of trypsin-digested peptides. The results are depicted in Then, we analyzed the phosphorylation status of HER1 Y1068 and of ERK1/2 T185 and Y187 following stimulation of 5637 or HeLa cells with 25 ng/mL HB-EGF or 200 ng/mL CXCL12 for 20 minutes. via PKC and a late one that was β-arrestin-dependent. By using Ro-31, which inhibits PKC, recombinant CXCL12 induced only a late, β-arrestin-dependent ERK1/2 phosphorylation spike, but [N33A]CXCL12 induced no ERK1/2 phosphorylation at all. Therefore, [N33A]CXCL12 did not induce the β-arrestin-dependent phosphorylation of ERK1/2, while eliciting the G-protein-dependent spike. Panels E and F in via PKC.If the variant form [N33A]CXCL12 induces full inhibition of HER1 for lack of β-arrestin activation, then recombinant CXCL12 should induce a full inhibition of HER1 in cells knockdown of β-arrestin. Therefore, to further test the transduction pathways downstream of CXCR4 we established a siRNA sequence targeted against the CXCR4-associated β-arrestin 2. This siRNA efficiently blocked β-arrestin in both 5637 and HeLa cells. 2+ flux, thus suggesting a mechanism by which known signaling participants can modulate HER1 signaling. After treatment with CXCL12 or [N33A]CXCL12, we observed that not only the PKC inhibitor Ro-31 . CsA, a specific inhibitor of calcineurin, prevented the CXCL12 effect and was capable of restoring the normal pattern of HER1 phosphorylation  activation of calmodulin/calcineurin and 2) transinhibition of tyrosine phosphorylation at the cytoplasmic tail of HER1. Prestimulation of HER1-positive target cells with CXCL12 modified HB-EGF-dependent HER1 stimulation by delaying peak phosphorylation at tyrosine 1068 and 1173. This effect was ultimately mediated by the CaWe demonstrate that in two different human neoplastic cell lines. But other studies provide circumstantial evidence that support and expand our findings, which are in line with experimental data regarding the effect of W-13 on HER1 activation via engineered CXCL12 with inhibitory capabilities.The CXCL12-mediated, calcineurin-dependent modulation of HER1 phosphorylation and the differential activity of recombinant and mutated molecules should be seen in the context of CXCR4 signaling specificity The modulation of HER1 phosphorylation may play a role in cellular responses characteristic of invasive growth. This can be thought of as a behavioral program that regulates cell dissociation from its microenvironment, thus promoting cell migration and the colonization of new sites. This program is shared by normal cells involved in developmental, inflammatory or immune tasks and cancer cells in the metastatic process. Based on the findings in the present and other works via CXCR4 is capable of activating an inhibitory calmodulin/calcineurin module that modifies the magnitude and persistence of HER1 signaling. Taken together, these data contribute a refinement to our understanding of the signaling downstream of CXCR4 and HER1 In conclusion, this work provides new evidence that the signaling mediated by CXCL12