G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC In addition to these soluble factors, platelets express a number of immunoglobulin (Ig)-like receptors at the platelet surface that relay a negative signal to the cell to either maintain the resting state or limit the extent of activation. These receptors elicit their effects via the immunoreceptor tyrosine-based inhibitory motifs (ITIM) or immunoreceptor tyrosine-based switch motifs (ITSM) in their intracellular tails, which can interact with Src homology (SH) 2 domain-containing proteins. There are a number of Ig-like ITIM/ITSM receptors expressed on the platelet surface, including G6B-b, platelet endothelial cell adhesion molecule-1 (PECAM-1), carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) and TREM-like transcript-1 (TLT-1).Platelets are essential for primary haemostasis, but also play a major role in the development of cardiovascular disease. Platelets are regulated by both activating and inhibitory signals and the balance of these opposing signals regulates the extent of cell activation and thrombus formation. Healthy endothelium releases soluble factors, such as prostacyclin . Streptactin-Sepharose beads were purchased from GE Lifesciences . The cell permeant SKF inhibitor, PP2, and its inactive analogue, PP3, were purchased from Merck Biosciences. The G6B monoclonal antibody has been previously described 2 was added before centrifugation at 1000 g for 10 minutes at room temperature. Platelets were resuspended in 1 ml Tyrodes buffer  pre-warmed to 30°C and 150 µl ACD. The volume was adjusted to 25 ml with Tyrodes buffer, followed by 3 ml ACD and 1.25 µg PGI2. Cells were centrifuged at 1000 g for 10 minutes at room temperature and the resultant cell pellet was resuspended in 1 ml pre-warmed Tyrodes buffer before adjusting the volume to a final cell density of 4×108 cells/ml (aggregations) or 2×109 cells/ml (peptide pull downs). Where appropriate, 1 mM EGTA, 10 µM indomethacin and 2 U/ml apyrase were added to inhibit aggregations (referred to as non-aggregating conditions).Whole blood was taken from healthy volunteers and collected into 50 ml syringes containing 5 ml acid citrate-dextrose (ACD) in accordance with procedures approved by the Local Research Ethics Committee (Ref: 07/Q1603/17). Platelet-rich plasma (PRP) was isolated by centrifugation at 200 g for 10 minutes at room temperature. PRP was pooled and 10 µg PGI9 cells/ml under non-aggregating conditions . Cells were activated with 1 ug/ml CRP for 90 seconds under stirring, but non-aggregating conditions, before reactions were terminated by the addition of 500 µl 2× NP40 lysis buffer  with inhibitors  and placed on ice. Lysates were pooled and pre-cleared as a batch with 200 µl streptactin beads (50% slurry). Non-specific interactions were then removed by centrifugation at 4,750 g for 15 minutes at 4°C. Pre-cleared lysates were incubated with 50 µl peptide/bead conjugate (50% slurry) at 4°C for 30 minutes with end-over-end mixing. The pre-cleared lysate was divided equally among the samples so that peptide-protein complexes were isolated from the same starting material. Bead/protein precipitates were washed 4 times in 1× NP40 lysis buffer and recovered by boiling in reducing Laemmli buffer prior to separation by SDS-PAGE under denaturing conditions.Forty micrograms of peptide were coupled to 50 µl of Streptactin beads (50% slurry) for 30 minutes at 4°C with end-over-end mixing, washed twice with 1 ml PBS and resuspended in 100 µl of modified Tyrodes buffer. Platelets were resuspended in modified Tyrodes buffer at a concentration of 2×108 platelets in a final volume of 100 µl DP lysis buffer. Samples were mixed on a rotating wheel at 4°C for 1 hour. Samples were washed once in 200 µl DP lysis buffer and twice in 1 ml PBS prior to boiling in Laemmli buffer for 5 minutes.One microgram of antibody was bound to 10 µl of Protein A-sepharose (20 µl of a 50% slurry), in a final volume of 100 µl PBS, by incubation on a rotating wheel for 90 minutes at 4°C. Washed human platelets were incubated with either Tyrodes, 5 µM PP2, 5 µM PP3 or pervanadate for 10 minutes, after which they were pelleted and lysed in DP lysis buffer  and 5% w/v n-Dodecyl-β-D-maltoside ) such that each IP sample received 4×10Proteins were separated by SDS-PAGE on pre-cast 4–12% NuPage polyacrylamide gels (Invitrogen) under denaturing conditions and transferred onto nitrocellulose membrane. Membranes were blocked with 5% Marvel/TBS/0.1% Tween-20 for 1 hour at room temperature. Primary and secondary antibody HRP conjugates were used according to the manufacturers' instructions. Bands were visualised with ECL Plus reagent .www.omc.ox.ac.uk] construct #3915), SHP-1 (N+C)SH2 domains  (OMC #3908M), SHP-2 (N+C)SH2 domains (His-tag) (3911), SHP-1 (N)SH2 (His-tag) (OMC #3909), SHP-1 (C)SH2 (His-tag) (OMC #3910), SHP-2 (N)SH2 (His-tag) (OMC #3912) and SHP-2 (C)SH2 (His-tag) (OMC #3913). These proteins were expressed in BL21(DE3) cells (Invitrogen) induced with 1 mM IPTG for 4 hours at 37°C. Bacteria were pelleted and washed in PBS/0.5 M NaCl prior to sonication in PBS/0.5 M NaCl, in the presence of protease inhibitor cocktail . Soluble and insoluble fractions were separated by centrifugation at 30,000 g for 60 minutes. Soluble fractions containing tagged proteins were incubated for 1 hour at 4°C with end-over-end mixing in the presence of washed Ni-Sepharose  supplemented with 40 mM imidazole or Amylose resin . Proteins were eluted according to the manufacturer's instructions and purified by size exclusion chromatography on an AKTApurifier .Constructs containing SHIP-1 SH2(His-tag)   and incubated with 100 pM of either SHP-1 SH2 MBP or SHP2 SH2 His protein in a total volume of 250 µl PBS for 30 minutes with end-over-end mixing at room temperature. Beads and supernatant were separated and the beads resuspended in a volume of PBS equal to the supernatant fraction. Equal volumes were loaded onto a 4–12% NuPage gel (Invitrogen) and separated by SDS-PAGE under denaturing conditions. Gels were stained with SimplyBlue Coomassie Stain (Invitrogen). For the single SH2 domain assays, 500 pM peptide was coupled to 25 µl Streptactin beads and incubated with 500 pM purified SH2 domain as described above.D was defined as the concentration of SH2 protein that gave half maximal binding to the peptide. For the 60-mer peptides, SH2 domains were passed over the chip surface at 50 µl/min. KD was determined as Koff/Kon, which were determined using both GraphPad Prism 5.0  and BIAevaluation Software .Experiments were conducted on a BIAcore™ 3000 in HBS buffer . Streptavidin was coupled to a research grade CM5 chip  as previously described GRKKRRQRRRPQDLDQEPSLLY*ADLDHLALSRPRRLSTADPADASTIY*AVVV or unphosphorylated control peptide (lacking tyrosine phosphorylation) - FLUO-GRKKRRQRRRPQDLDQEPSLLYADLDHLALSRPRRLSTADPADASTIYAVVV. TAT sequences are underlined and phosphorylated tyrosines marked with an asterisk. Peptides were loaded into washed platelets by incubation with 10 µM peptide for 15 minutes at 30°C, after which they were assessed by light transmission aggregometry. Peptide loading conditions were optimised and verified by flow cytometry.Fluorescein-labelled, TAT-G6B-b peptides were synthesised by Peptide Protein Research Ltd . Peptides were either unphosphorylated, or phosphorylated at both ITIMs as follows: Dual phosphorylated peptide – FLUO-SHP-1 has been shown to associate with G6B-b in human platelets. SHP-2 has also been demonstrated to associate with G6B-b, but this has not been demonstrated in human platelets. We wanted to determine whether SHP-2 could associate with the receptor in human platelets as this interaction could relate to receptor function and regulation of GPVI signalling. As G6B-b is an orphan receptor, it is not possible to activate the receptor with its endogenous ligand. In the absence of receptor agonists, we carried out co-immunoprecipitation experiments in platelet lysates treated with the SFK inhibitor, PP2, its non-active analogue, PP3, or the tyrosine phosphatase inhibitor, pervanadate. This generated samples with G6B-b in a state of non-phosphorylation, partial/sub-maximal, or maximal phosphorylation, respectively . Under tPrevious mutagenesis experiments in Cos7 cells demonstrated that association of SHP-1 and SHP-2 with G6B-b relies on phosphorylation of the ITIM/ITSMs. Although likely that these interactions are direct, this has not yet been conclusively shown. In the absence of an endogenous ligand or receptor agonist, we used phosphopeptides corresponding to the intracellular portion of G6B-b in which the ITIM and ITSM reside. These biotinylated peptides were phosphorylated at either the ITIM, the ITSM, or at both sequences, to mimic receptor activation and phosphorylation. These peptides were incubated with platelet lysate and isolated using Streptacin beads. Peptide associated material was separated by SDS-PAGE and assessed for the presence of SHP-1 and SHP-2 by Western blotting, both of which were clearly identified . PeptideInteractions between SH2 domains and ITAMs, ITIMs and ITSMs often rely on direct association between the phosphomotif-bearing receptor and its SH2 domain-containing binding partner. We wanted to verify that G6B-b was associating directly with SHP-1 and SHP-2 and not via an intermediary. To address this, we incubated G6B-b peptides with equimolar quantities of purified SH2 domain (either N or C or N+C) from SHP-1 and SHP-2. This approach revealed a robust association between SHP-2 and G6B-b phosphopeptides  with ∼70It is known that SH2 domains exhibit preferential interactions with different consensus phosphotyrosine sequences largely determined by the amino acids immediately downstream of the central phosphotyrosine D of 130 nM (single site fitting). The pY211 peptide bound SHP-2 N(SH2) with a KD of 110 nM (single site fitting). This was determined by plotting equilibrium binding  is most important for receptor-phosphatase association with the second site playing a minor, but critical role. Previous studies have shown that using single phosphomotifs (isolated from sequences in which they may exist in tandem) and single SH2 domains can be misleading D = koff/kon) by SPR at physiological temperatures. The presence of both phosphomotifs dramatically enhanced association of SHP-1 tandem (N+C) SH2 domains to the dual phosphopeptide. Single site models were found to fit the data well while bivalent analyte models did not suggesting that SHP-1 tandem SH2 domains bind to the G6B-b dual phosphopeptide as a single binding unit, rather than as a two-site association, with an affinity of 35.3±4.3 nM , with a KD of 0.48±0.09 nM  was observed , therefore reducing platelet sensitivity to agonist. Effects on the endogenous GPVI receptor ligand, collagen, were more pronounced (50 from 0.4 µg/ml to 1 µg/ml. Thrombin-mediated activation was unaffected (data not shown). Increasing the concentration of G6B-b dual phosphorylated peptide inside the cell reduces platelet sensitivity to GPVI agonists. Peptide loading was verified by flow cytometry , SHP-2 has a more rapid association rate, resulting in a higher affinity for G6B-b than SHP-1. SHP-2 tandem SH2 domains bind to the G6B-b dual phosphopeptide with an affinity (KD = 0.48±0.09 nM) similar to that previously reported for an optimal SHP-2 binding protein, SHPS-1/SIRPα d of ∼1 µM) and SHP-2 (Kd = 0.5 nM), independently. Although the G6B-b/p85 interaction is much lower than the nanomolar binding affinities documented for other p85-binding proteins, SPR showed this interaction to be direct and within physiological limits for SH2-pY associations.Platelet activation is a tightly regulated process, being influenced by a combination of both positive and negative signals. The ITIM/ITSM-bearing receptors PECAM-1, CEACAM1L and G6B-b can all inhibit platelet function and have been shown to associate with SH2 domain-containing phosphatases. Our data revealed differences in the binding properties of SHP-1 and SHP-2 for G6B-b. While both phosphatases have the same dissociation rate for the dual phosphorylated peptide  study. Although the affinity for activating binding partners is weak, G6B-b is present at a high copy number at the cell surface In this study we also detected a number of other SH2 domain-containing proteins in phosphopeptide-associated material. In addition to the p85 regulatory subunit of PI3K, Syk and PLCγ2 were found to weakly associate with endogenous G6B-b in pervanadate-treated cells, but these interactions appear somewhat tenuous and secondary to the interactions of G6B-b with its dominant binding partners, SHP-1 and SHP-2. Mori 50 of GPVI agonists. It is also possible that the presence of G6B-b phosphopeptide would compete for SHP-1 and SHP-2 binding, impeding their recruitment to the membrane. If SHP-1 and SHP-2 have inhibitory roles, one might expect that their loss from signalling hubs close to the membrane would increase cell sensitivity to agonist but this does not appear to be the case. The lack of an endogenous ligand for G6B-b has been limiting with regards to elucidating its role in cell activation and function, and it is therefore difficult to progress this work and fully characterise the receptor. Despite this, we have identified a number of alternative binding partners for G6B-b that may shed light on the work by Mori et alIt is likely that G6B-b contributes to platelet regulation by employing both passive (via interactions with activating proteins such as Syk and PI3K to prevent spurious cell activation) and active (recruitment of SHP-1 and SHP-2 to the membrane) mechanisms. One might envisage a situation where in the resting cell, G6B-b is able to associate with any number of SH2 domain-containing proteins, including Syk, PLCγ2 and p85, such that it can act as a sink, or buffer, for these proteins. Once the cell is activated by collagen, FcRγ will be phosphorylated, generating a high affinity binding site for Syk, which will then predominantly associate with the ITAM to initiate cell signalling. This then leads to LAT phosphorylation and subsequent recruitment of PI3K. Although this hypothesis is highly speculative, some evidence to support this theory is presented in the peptide-loading experiments described above. Loading dual phosphorylated peptide into washed human platelets reduces cell sensitivity to GPVI ligands; if G6B-b was acting as a ‘sink’ for activating SH2 domain-containing proteins, one would predict that an excess of phosphopeptide would increase the number of alternative binding sites for these proteins and therefore increase the ECin vivo and ensure that cell activation occurs only in the presence of an appropriate physiological stimulus.Receptor-ligand dynamics will influence cell activity and are themselves influenced by copy number, localisation, phosphorylation and binding affinity. The combination of all these factors will serve to buffer spurious cell signalling events that could lead to inappropriate platelet activation and thrombus formation


Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade. Signal transduction pathways and networks direct cell responses largely through post-translational modifications, e.g., phosphorylation/dephosphorylation of their protein components. But the rates of these modifications depend in turn on the intracellular concentrations of enzymes and other regulatory proteins; thus, mechanisms governing protein synthesis and degradation are equally central to the regulation of cell signaling.The ubiquitin-proteasome pathway is an essential quality control mechanism directing degradation of mislocated, misfolded, and damaged proteins, and, by tempering the expression levels of specific signaling proteins, it also exerts a level of control over cell physiology The mitogen-activated protein kinases (MAPKs) ERK2/MAPK1 and ERK1/MAPK3 (hereafter referred to collectively as ERK1/2) are activated by phosphorylation in a canonical Raf → MEK → ERK kinase cascade in response to most growth factors and cytokines, and ERK1/2 phosphorylate more than 150 cytosolic and nuclear substrates Considering that a host of intracellular proteins are affected by proteasome inhibition, coupled with evidence that knockdown of MKP3 expression enhances growth factor-stimulated ERK phosphorylation in some contexts 751, Akt pSer473, ERK pThr202/pTyr204, and MEK pSer217/pSer221 were from Cell Signaling Technology . Antibodies against MKP1 were from Santa Cruz Biotechnology . MG132 was purchased from Calbiochem  and aliquoted in DMSO; cells were incubated with the drug at a final concentration of 25 µM, with an equivalent concentration of DMSO (0.2% v/v) serving as a vehicle control. All tissue culture reagents were from Invitrogen . Unless otherwise noted, all other reagents were from Sigma-Aldrich .Human recombinant PDGF-BB and murine recombinant FGF-2 were purchased from Peprotech . Antibodies against total ERK1/2, MEK1/2, Akt1/2/3 and MKP3 and phospho-specific antibodies against PDGF β-receptor pTyr2 in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and the antibiotics penicillin and streptomycin. Cells were serum-starved for 3 hours, followed by pretreatment with MG132 or DMSO vehicle control for the time indicated. The cells were then stimulated with either PDGF-BB or FGF-2 as indicated, in the continued presence of MG132 or DMSO. Quantitative immunoblotting from detergent prepared lysates was performed using enhanced chemiluminescence, and densitometry data were normalized as described in detail previously NIH 3T3 mouse fibroblast and HT-1080 human fibrosarcoma cell lines were acquired from American Type Culture Collection . Mouse embryonic fibroblasts, derived from pregnant CD-1 mice , were isolated according to standard protocol A semi-mechanistic model of ERK phosphorylation was developed to estimate the fold-upregulation of ERK phosphatase activity in MG132-treated cells, using the time course of MEK phosphorylation as an input. Given that MEK phosphorylation is also perturbed by MG132 treatment, our strategy was to independently fit each time course of MEK phosphorylation to a phenomenological function; then, assuming those phosphorylated MEK kinetics, ERK phosphorylation kinetics were globally fit to a modified Michaelis-Menten model .n = 104) ensemble of “good” parameter sets rather than one “best” fit. After compiling the ensemble, the model output is recalculated for each parameter set, and at each time point, an ensemble mean and standard deviation are calculated.All calculations were performed using MATLAB . The parameter estimation approach used is as described in detail previously 751 of PDGF β-receptor, a major phosphorylation site that contributes to the recruitment of phosphoinositide 3-kinase (PI3K), in a PDGF dose-dependent manner  that produce nearly equivalent qualities of fit, allowing us to evaluate the degree to which each parameter was properly constrained (This model was iteratively fit to the ERK data set by Monte Carlo sampling of the model parameters to obtain a large ensemble of parameter sets (strained . As a cestrained . The corp = 0.05 level (two-way ANOVA); this is attributed to the shape of the MKP3 time course, which dips down at early stimulation times, bringing MKP3 expression in MG132-treated cells down to a level that is similar to those in control cells at time zero and at time = 120 minutes  and HT-1080 human fibrosarcoma cells . In the Pharmacological inhibitors vary in both promiscuity and breadth of biological outcomes. An inhibitor might antagonize multiple molecular targets, or it might act on a quite narrow range of targets that nonetheless mediate pleiotropic effects. Broad effects should be expected in cells treated with a proteasome inhibitor, irrespective of its specificity. The ubiquitin-proteasome degradation pathway clearly shows some degree of selectivity in regulating the expression levels of protein targets, but it nonetheless impacts a broad range of signal transduction pathways and other intracellular processes.751 was reduced, as were the phosphorylation levels of the downstream kinases Akt, MEK, and ERK. A plausible explanation for these findings is that multiple phosphatases are upregulated in proteasome-inhibited cells. In endothelial cells, proteasome inhibition has been shown to upregulate the serine-threonine phophatase PP2A, accompanied by reduced Akt phosphorylation 751 phosphorylation at the low PDGF dose  of MEK on ERK is directly proportional to the measured level of phosphorylated MEK; in turn, this allowed the evaluation of the postulated upregulation of ERK phosphatase activity. In contrast, it was not prudent to attempt to model in mechanistic detail the multiple effects of proteasome inhibition affecting the kinetics of MEK phosphorylation. Thus, consideration of the mechanistic uncertainties in constructing such a mathematical model can serve as a guide as to which research questions might be addressed given the data in hand.The complexity of ERK modulation by proteasome inhibition, considering the direct and indirect effects on ERK phosphorylation status and the dynamic nature of the pathway, demands a quantitative analysis. We contend that kinetic modeling is a useful approach for parsing multiple, time-dependent effects on biochemical systems. A key step in its implementation is choosing the degree of model complexity, since the mathematical description of a system’s mechanistic details comes with the need to specify a certain number of rate parameters, which might or might not be appropriate depending on the availability of quantitative data Text S1Details of Computational Methods.(PDF)Click here for additional data file.


Competition is ubiquitous among social animals. Vying against a conspecific to achieve a particular outcome often requires one to act aggressively, but this is a costly and inherently risky behavior. So why do we aggressively compete, or at the extreme, fight against others? Early work suggested that competitive aggression might stem from an innate aggressive tendency, emanating from subcortical structures. Later work highlighted key cortical regions that contribute toward an instrumental aggression network, one that is recruited or suppressed as needed to achieve a goal. Recent neuroimaging work hints that competitive aggression is upmost a cost-benefit decision, in that it appears to recruit many components of traditional, non-social decision-making networks. This review provides a historical glimpse into the neuroscience of competitive aggression, and proposes a conceptual advancement for studying competitive behavior by outlining how utility calculations of contested-for resources are skewed, pre- and post-competition. A basic multi-factorial model of utility assessment is proposed to account for competitive endowment effects that stem from the presence of peers, peer salience and disposition, and the tactical effort required for victory. In part, competitive aggression is a learned behavior that should only be repeated if positive outcomes are achieved. However, due to skewed utility assessments, deviations of associative learning occur. Hence truly careful cost-benefit analysis is warranted before choosing to vie against another. A critical consideration in social decision-making is whether or not to compete against a conspecific. Competitive action can take many forms, for example it could involve a quick, direct, physical fight between two individuals, or long, covert, strategic manoeuvres between groups. In all of its forms, competitive engagement carries the implicit goal of outperforming conspecifics in order to achieve resources or other outcomes that facilitate self-preservation. Direct competitive aggression is one of the most observable forms of competitive engagement amongst social animals. This specific form of competitive action is frequently required to obtain or protect a resource, but it is energetically costly and inherently risky. So how do we know when (or when not) to put up a fight?Fight Club, it does not fit well with common patterns of animal behavior. Socially, constant aggressive tendencies would create a tense and nihilistic world. Physiologically, subcortical circuits that were basally active would require an inordinate amount of cortical energy to suppress. It would be more evolutionarily advantageous for animals to have an instrumental aggression network (IAN), one that can be recruited for competitive action only when it's worthwhile to compete.Early investigations in psychoanalysis, ethology and neuroscience suggested that animals have an innate aggressive drive, stemming from basally active subcortical networks. While this idea might explain the behavior of certain characters from the 1990's media phenomenon Determining whether competitive aggression is worthwhile represents a cost-benefit decision, largely reliant on the same neural networks that process non-social decision variables. There is an outcome at stake that you want. How much do you want it and what costs will be incurred in obtainment? Reward valuation and cost assessment are paramount. The presence of others who also want that same outcome simply makes for multi-factorial cost-benefit analysis. Peer interest should enhance the utility of the outcome, providing an endowment effect that can be modulated by the composition of the peer group, and the expected ferocity of their competitive tactics. Expended competitive effort can discount the utility of the outcome, but can also provide an immediate endowment effect of deservingness for the victor. Multi-factorial cost-benefit analysis thus structures competitive aggression, informing us when and when not to put up a fight.thanatos  can elicit aggressive behaviors, vocalizations and lowered fear responses in a variety of species  increases hypothalamic excitability while stimulation of the corticomedial amygdala (CMA) suppresses hypothalamic discharge , which near-reflexively activate the sympathetic nervous system and the thalamo-amygdala pathway. This can prompt IAN activation and subsequent aggression. Indeed aggression is frequently observed in response to painful stimuli, providing a feedforward mechanism for escalation of aggression in combative fights.Alternatively, this CMA braking mechanism on the IAN may be potentiated by fear- or caution-inducing stimuli , which would contribute toward the efficacy of threat cues in preventing competitive fights. In rats, CMA lesion results in failure to avoid dominant conspecifics , aggressively (contest competition) or slyly (strategic competition). In line with this idea, abnormalities in amygadalar activity would manifest as impaired competitive effort allocation, generating a spectrum of behaviors ranging from hyperaggression on one end, to avolition and social withdrawal on the other. A similar spectrum is seen following damage to regions of the prefrontal cortex (PFC). Blumer and Benson's characterization , OFC, anterior cingulate cortex (ACC) and dlPFC have been implicated in controlling aggressive behaviors. Prefrontal regulatory control over the IAN can occur via direct pathways to the subcortical nuclei or via indirect pathways utilizing the amygdala  is associated with PFC activation in humans and rats . Win-related TPJ activation is greater when a larger reward is at stake  of x; x being a value greater than zero, representative of a cost-benefit valuation that has been previously established via trial-and-error and/or observational learning. Chang et al. (x) based on frame of reference. Self-referenced valuations predominate in the OFC and ACC sulcus, with the former sensitive to self-experienced rewards and the latter to self-experienced foregone rewards. Other-referenced valuations predominate in the ACC gyrus , where a1 represents peer interest endowment and ranges from 0 to 1. No peer interest in the resource (a1 = 0), up to high peer interest in the resource (a1 = 1) modulates the perceived utility of the resource which can prompt action. Alternatively, if peer disgust is exhibited for the resource, −1< a1 < 0, decreasing utility and dissuading action.In a competitive social scenario, the resource still holds a value of a1) of the monetary value (x). Likewise, Azzi et al. (U = x), the mere presence of a conspecific effectively doubles single-unit encoding of reward value in the OFC, perhaps reflective of U = x + (x * a1).Behaviorally, the mere presence of others does prompt resource scavenging, as is seen in social facilitation of feeding. Satiated animals will start eating again when new animals arrive and start eating Bayer, . Rats tri et al.  have reca1) is further modified by the composition of the peer group  and their anticipated aggressiveness , such that U = x + (x * (a1 * (g * y))). A congenial group of competitors who also want the resource (g = 1) would exert no further change in utility beyond the initial peer endowment effect (a1). A group of established adversaries who also want the resource (g = 2) would effectively double the peer endowment effect, trebling utility from the initial intrinsic value x. However, even if a resource has become highly valuable due to an adversary's interest in the resource, impulsive action would be unwise without weighing in potential losses that might soon occur, in terms of lost effort, lost status or even loss of life. If anticipated competition is expected to be fair, with acceptable, proportional costs, y = 1. However, if anticipated competition is expected to be highly aggressive and contentious, for example against an established dominant conspecific, y = −1. Hence utility assessments would be highest for a fair fight against adversaries, and lowest for an anticipated unfair/hostile fight against adversaries.Peer interest endowment  prior to competitive encounters, as these regions are sensitive to conspecific assessment in non-competitive tasks. For example, in a human imaging study by Behrens et al.  need to be assessed and integrated with resource information (x) to guide choice behavior. The higher the utility estimate that results from U = x + (x * (a1 * (g * y))), the higher the likelihood that a subject will choose to engage in competition.The ACC, vmPFC, and OFC also likely contribute toward peer-related valuations , and one from continued peer interest (a2). Effort itself is generally aversive and can discount initial estimates of x, affecting choice behavior  should therefore enhance perceived utility immediately after the contested-for outcome has been achieved: U = x + (x * (a1 * (g * y))) + e. When effort is required to obtain a reward, BOLD activity increases in the amygdala and striatum upon reward receipt, while OFC reward-related activity remains unchanged , a subtle schadenfreude type endowment effect. Continued peer interest endowment (a2) may be modified by peer group composition (1 < g < 2), similar to what is proposed for a1, whereby U = x + (x * (a1 * (g * y))) + e + (a2 * g). Schadenfreude and its opposing partner envy are more likely to arise when fellow competitors (g) are self-relevant, salient conspecifics )) + e + (a2 * g). The joy of winning and pain of losing have recently been posited as single variables ρwin and ρloss by van den Bos et al. ))] and in the outcome evaluation phase [U = x + (x * (a1 * (g * y))) + e + (a2 * g)]. These skewed utility estimates can prompt competitive actions which are ultimately costly to the individual or group. Peer group endowment effects may be particularly strong in adolescence (Blakemore and Robbins, In part competitive aggression is a learned mode of behavior, repeated when it is reinforced. But it is also a behavior motivated by skewed utility estimates, which may account for some of the violations of associative learning that are commonly observed in competitive environments. A preliminary model has been posed herein to illustrate how, in competitive scenarios, peer group endowment effects act to artificially inflate perceived benefit of a resource in the decision phase [The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.001) with lithospermic acid (LSA), protocatechuic acid, rosmarinic acid (RMA), and salvianolic acid A (SAA) producing virtually complete inhibition under test conditions. Kinetic analysis demonstrated that LSA, RMA, and SAA were competitive inhibitors. As such, Ki values were estimated as 14.9 ± 4.9 μM for LSA, 5.5 ± 2.2 μM for RMA, and 4.9 ± 2.2 μM for SAA on mOat1-mediated transport, and as 31.1 ± 7.0 μM for LSA, 4.3 ± 0.2 μM for RMA, and 21.3 ± 7.7 μM for SAA on mOat3-mediated transport. These data suggest that herb-drug interactions may occur in vivo on the human orthologs of these transporters in situations of polypharmacy involving Danshen and clinical therapeutics known to be organic anion transporter substrates.Many active components of herbal products are small organic anions, and organic anion transporters were previously demonstrated to be a potential site of drug-drug interactions. In this study, we assessed the inhibitory effects of six hydrophilic components of the herbal medicine Danshen, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and tanshinol, on the function of the murine organic anion transporters, mOat1 and mOat3. All of Danshen components significantly inhibited mOat1- and mOat3-mediated substrate uptake ( Salvia miltiorrhiza), has been employed for thousands of years in the relief of symptoms of cardiovascular disease PAH) and estrone sulfate ([3H]ES) were purchased from PerkinElmer Life and Analytical Sciences  and unlabeled PAH, ES, and probenecid were purchased from Sigma-Aldrich .The Danshen components LSA, PCA, RMA, SAA, SAB, and TSL (≥96% purity) were obtained from Tauto Biotech . Their chemical structures are illustrated in 2 in DMEM F-12 media  containing 10% serum, 1% Pen/Strep, and 125 μg/mL hygromycin B.Derivation of the stably transfected Chinese hamster ovary (CHO) cell lines expressing mOat1 (CHO-mOat1), mOat3 (CHO-mOat3), and the empty vector (FRT) transfected control cell line (CHO-FRT) was described previously , 15. Cel5 cells/well were seeded in 24-well tissue culture plates and grown in the absence of antibiotics for 48 hr. On the day of the experiment, cells were equilibrated with transport buffer for 10 min . Equilibration buffer was replaced with 500 μL of fresh transport buffer containing 5 μM [3H]PAH or 1 μM [3H]ES (0.25 μCi/mL) with or without inhibitors. After incubation, cells were immediately rinsed three times with ice-cold transport buffer, lysed, and analyzed via liquid scintillation counting. Uptake values were normalized to corresponding total protein content in cell lysates as determined by the Bradford method. Substrate accumulation is reported as picomoles of substrate per milligram protein. Substrate concentration and accumulation time used for kinetic analysis of mOat3  were determined previously /Km) /Km) . Resultst-test or using Student's unpaired t-test, as indicated (α = 0.05).Data are reported as mean ± S.D. or mean ± S.E.M. as indicated. Statistical differences were assessed using one-way ANOVA followed by post hoc analysis with Dunnett's P < 0.001) under these conditions. LSA, SAB, and TSL produced approximately 70–85% inhibition, whereas PCA, RMA, and SAA, each reduced PAH accumulation to background level (>95% inhibition), similar to the prototypical OAT inhibitor, probenecid. Further, the addition of these compounds (1 mM) did not significantly influence the low, probenecid-insensitive  PAH uptake in the CHO-FRT cells (data not shown), indicating that the reduction in uptake of PAH in the CHO-mOat1 cells is attributable to the inhibition of mOat1 activity and that CHO-FRT PAH level serves as an appropriate background correction factor.Accumulation of PAH in the CHO-mOat1 cell line (98.5 ± 14.6 pmol/mg protein/10 min) was ~30 fold greater than that in the background control CHO-FRT cells . SAB and TSL produced approximately 53% and 55% inhibition, respectively. LSA, PCA, RMA, and SAA, like probenecid, blocked virtually all (>91%) mOat3-mediated ES transport (Stably transfected mOat3-expressing (CHO-mOat3) cells showed significantly greater accumulation of ES (~20 fold) relative to control CHO-FRT cells  values , inhibition of mOat1- or mOat3-mediated transport was measured  was >0.9. No significant differences between Ki values for mOat1 and mOat3 were detected as determined by Student's unpaired t-test.To allow direct comparison of inhibition potencies of LSA, RMA, and SAA for mOat1 and mOat3, experiments were conducted to determine the inhibition constant  identified to date [Aristolochia sp. plants, was identified as the causative agent for “Chinese herbs nephropathy” (now referred to as aristolochic acid nephropathy). When investigated, aristolochic acid was found to be a high-affinity substrate for both mOat1 (Km = 790 nM) and mOat3 (Km = 514 nM), suggesting that these transporters are key mediators in the renal proximal tubule cell accumulation of this toxicant [Many natural products, which are extracted from living organisms, have beneficial biological or pharmacological activities and have been used as medicines throughout the world for more than 1,000 years. Today, they are often used as first-line therapeutics, dietary supplements, or complementary/alternative medicines, and it is reported that ~20% of adults in the United States are taking an herbal product [ to date . Recentltoxicant , 22. Thuhttp://clinicaltrial.gov/ct2/show/NCT00797953?term=tasly&rank=1). To date, however, no formal studies examining the interaction between OATs and hydrophilic Danshen compounds, which are small organic acids and known to be major components in its pharmaceutical products, have been reported. Therefore, in the present study, we investigated whether any of the six purported active Danshen components, LSA, PCA, RMA, SAA, SAB, and TSL, interacted with the transporters mOat1 and/or mOat3  for LSA and SAA than mOat3. However, in general, all of the values are quite similar and exhibit well below an order of magnitude in difference. The estimates are comparable to those reported for the strong OAT inhibitor, probenecid, Ki = 6.4 and 4.6 μM for mOat1 and mOat3, respectively [A major advantage of this study is that while Danshen preparations  include multiple active components, purified preparations of individual Danshen extract components were used. Thus, the strength of interaction of each compound could be quantified independently versus the merged effects of a mixture containing all of the compounds. Similarly, since the transporters were expressed in isolation, the interaction of each individual compound with each individual transporter could be specifically assessed versus the  Figures . The rani ratio  suggestsectively , 23, 24.In vivo pharmacokinetic studies have indicated that elimination of hydrophilic Danshen components involves active renal secretory pathways. In rats, the unbound renal clearance of TSL was ~5X greater than the glomerular filtration rate [in vivo results may be explained by competition for transport on Oat1/Oat3. An in vivo pilot study conducted in healthy humans reported Cmax values for LSA and RMA of 2.1 μM and 6.7 μM after i.v. dosing of a Danshen product [Cmax values are ~0.07–1.56-fold higher than the Ki values for mOat1 and mOat3 determined in the present study (Cmax/IC50 (or Ki) ≥ 0.1, it indicates that drug-drug interactions may occur in vivo [Cmax/Ki ratio for RMA would be 0.12 and 0.16 on mOat1 and mOat3, respectively, indicating potential for herb-drug interactions. Naturally, if human OAT orthologs show similar (or higher) affinities, possible herb-drug interactions should be considered.ion rate . A separion rate . As we dnt study . As reco in vivo . Unfortu in vivo . HoweverA recent investigation indicated that flavonoid and hydroxycinnamic acid aglycones and their phase II metabolites  significantly inhibit hOAT1 and hOAT3 transport activity , 29. It  in vivo needs to be considered until investigated directly. For example, it was recently suggested that SAB might be an OATP substrate based upon inhibition of total and biliary clearance of SAB by rifampicin, an OATP inhibitor, in rats [In addition, there are other transporter families known to interact with small organic anions, for example, the multidrug resistance-associated proteins  and the organic anion transporting polypeptides . For the most part these latter two transporter families and the OATs have fairly distinct substrate interactions; however, the possibility that they also may contribute to the overall clinical pharmacokinetic profile of Danshen components in rats .Cmax of RMA after i.v. infusion of a 160 mg dose was 1.2–1.6-fold higher than the Ki values for mOat1 and mOat3 [Finally, the amount of each active component in Danshen preparations varies with different cultivation regions and manufacturing processes . The varnd mOat3 . These d


H2O2-mediated oxidative stress induced phosphorylation of caveolin-1 and association between p-caveolin-1 and SHP-2. SHP-2 specifically bound to wild-type caveolin-1 similarly to c-Src tyrosine kinase (CSK), but not to phosphorylation-deficient mutant of caveolin-1 (Y14A), and interfered with complex formation between caveolin-1 and CSK. In the presence of CSK siRNA, binding between caveolin-1 and SHP-2 was enhanced by H2O2 treatment, which led to reduced Src phosphorylation at tyrosine (Tyr) 530 and enhanced Src phosphorylation at Tyr 419. In contrast, siRNA targeting of SHP-2 facilitated H2O2-mediated interaction between caveolin-1 and CSK and enhanced Src phosphorylation at Tyr 530, leading to subsequent decrease in Src downstream signaling, such as focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). Our results collectively indicate that SHP-2 alters Src kinase activity by interfering with the complex formation between CSK and phosphotyrosine caveolin-1 in the presence of H2O2, thus functions as a positive regulator in Src signaling under oxidative stress in brain astrocytes.Reactive oxygen species (ROS) regulate diverse cellular functions by triggering signal transduction events, such as Src and mitogen-activated protein (MAP) kinases. Here, we report the role of caveolin-1 and Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) in H After 2–3 weeks of culture, the flasks were shaken to detach microglia and oligodendrocytes growing on the top of the astrocytic layer. The remaining adherent astrocytes were detached using trypsin/EDTA and plated on dishes. Microglia were detached from flasks by mild shaking and filtered through a nylon mesh to remove the astrocytes. The cell identities in astrocyte- and microglia-enriched cultures were confirmed by staining for glial fibrillary acidic protein.Human astroglioma CRT-MG, U87-MG, and U251-MG cells 2O2, N-acetyl-l-cysteine (NAC), and catalase were purchased from Sigma . PTP inhibitor IV (PTPi IV) was purchased from Calbiochem . Recombinant glutathione S-transferase (GST)-SHP-2 and GST-CSK were purchased from Abnova . Antibodies against caveolin-1, p-caveolin-1 (pY14) and SHP-2 were obtained from BD Transduction Laboratories . Anti-CSK antibody and anti-FAK were obtained from Santa Cruz Biotechnology . p-SHP-2 Y542, p-SHP-2 Y580, Src, p-Src Y416, p-Src Y529, and p-FAK antibodies were obtained from Cell Signaling . Tubulin antibody was obtained from Sigma. Horseradish peroxidase (HRP)-conjugated secondary antibodies for immunoblotting were obtained from Santa Cruz Biotechnology.HPfu buffer, Pfu turbo DNA polymerase and 125 ng of each of the mutagenic primers: sense, 5′-tcggagggacatctcgccaccgttcccatccg-3′ and antisense, 5′-cggatgggaacggtggcgagatgtccctccga-3′. Samples were then incubated at 95°C for 1 min, then for 20 cycles of 95°C for 1 min, 65°C for 4 min, and 68°C for 12 min. The methylated parental DNA template in the PCR product was digested using the DpnI (10 U) restriction enzyme for 3 h at 37°C. The reaction mixture was then directly transformed into XL1 Blue supercompetent Escherichia coli cells (Stratagene) and plated onto LB agar plates containing ampicillin. DNA was extracted from selected clones using the Labopass™ Gel Extraction Kit  and sequenced to verify the presence of the desired mutation. After mutagenesis, 1 µg of caveolin-1 wild-type (WT) or Y14A mutant DNA was transfected into HEK 293T cells using VIVAMAGIC transfection reagent  containing 10% FBS according to the manufacturer’s instructions for 24 h before treatment with 5 mM H2O2 for 10 min; the cells were then analyzed by immunoblotting.cDNA encoding full-length human caveolin-1 was obtained from Thermo Scientific . The Y14A caveolin-1 mutant was generated using a QuickChange site-directed mutagenesis kit  using PCR. pCMV-SPORT6-caveolin-1 cDNA (25 ng) was used as a template and added to dNTP mix, 2O2 for 10 min and were then analyzed by immunoblotting.Cells were plated and transfected with green fluorescence protein (GFP) siRNA  as a control group and human CSK siRNA and human SHP-2 siRNA from Bioneer  using Lipofectamine RNAiMAX  according to the manufacturer’s instructions to achieve a final concentration of 300 nM. Cells were allowed to recover in DMEM containing 1% FBS for 48 h before treatment with 5 mM HCells were washed with phosphate-buffered saline (PBS) followed by lysis in cold radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors  for 1 h on ice. Total lysates (700 µg) were pre-cleared by incubating with protein A-agarose beads  for 3 h at 4°C. After centrifugation, cleared lysates were incubated with 1 µg of anti-caveolin-1, anti-SHP-2, and anti-CSK antibodies at 4°C overnight, and precipitated with protein A-agarose beads for 3 h at 4°C. Immunoprecipitated proteins were separated by 10% or 12% SDS-PAGE, and input proteins were analyzed by immunoblotting as described.g) at 4°C for 30 min and resolved by 10% or 12% SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membranes , and then blots were probed with caveolin-1, p-caveolin-1, SHP-2, p-SHP-2, CSK, Src, p-Src, ERK, p-ERK, FAK, p-FAK and tubulin antibodies. After incubating with specific secondary antibodies, the blots were exposed to blue X-ray film  by using an enhanced chemiluminescence system .Cells were lysed in ice-cold RIPA buffer containing protease inhibitors for 1 h on ice. Lysates were centrifuged  Kit . Cells were plated and treated with or without 5 mM H2O2 for 10 min, fixed with 100% methanol at −20°C for 30 min, and then incubated overnight with antibodies against caveolin-1 and SHP-2 at 1∶100 dilution. After washing, the PLA probe PLUS against SHP-2 and PLA probemaker probe against caveolin-1 were incubated for 3 h at 37°C and the ligases (1 U/µL) diluted at 1∶40 were incubated for 2 h at 37°C. Circular oligonucleotides were amplified using polymerase (10 U/µL) diluted at 1∶80 for 100 min at 37°C. Samples were viewed using a Carl Zeiss confocal microscope . Nuclei were stained with VECTASHIELD HardSet™ mounting medium with DAPI . Experiments were repeated 4 times and showed similar results.Protein-protein interactions were detected using the Duolink II Identification of binding sites for recombinant SHP-2 and CSK in the caveolin-1 protein was performed using PepSPOTs . Synthetic peptide sequences of caveolin-1 (1–85 and 1–28) were used in this study. The peptide library contained either 13-mer peptides that overlapped by 5 residues or 13-mer peptides that overlapped by 8 residues. Each spot carried approximately 5 nmol of peptide covalently bound to the cellulose-betaalanine-membrane (N-terminus: acetyl). Two identical membranes were incubated overnight with either recombinant GST-SHP-2 (rGST-SHP-2) or GST-CSK (rGST-CSK) respectively, and immunoblotting was performed to detect rGST-SHP-2/caveolin-1 peptide and rGST-CSK/caveolin-1 peptide interactions.2 gas. Recombinant GST-SHP-2 or GST-CSK was mixed with 20% ethylene glycol and then spotted onto the chip. Ligands were reacted on room temperature for 12 h to allow the ligands to become fixed onto the chip. After the reaction, the ligands were removed by washing with 1×Tris-buffered saline (TBS), and purged with N2 gas. SPR signal baselines were stabilized using 1×TBS running buffer, and different dilutions of caveolin-1 phosphopeptides  in buffer were injected at a flow rate of 20 µL/min. Response unit (RU) alterations following sample reactions were analyzed by comparing affinities to the chip surface. Experiments were conducted by switching from samples immobilized on the chip (Cav-1 pY 14) to injected samples (rGST-CSK or rGST-SHP-2). Experiments were repeated twice and showed similar results. Equilibrium binding curves and RU values are shown.SPR was performed on a SPRmicro system . A G1 chip (bare surface gold chip) was dipped into a 3 mM N-hydroxylsuccinimide self-assembled monolayer to form covalent bonds among protein amine groups. After washing with ethanol, the chip was purged with NEscherichia coli (BL21) as soluble. Briefly, the transformed bacteria were grown in LB medium with 0.1 mg/ml ampicillin at 37°C to an A600 of 0.6, and then cultured for an additional overnight at 27°C after being induced with 0.5 mM IPTG . The cells were harvested by centrifugation at 8000 g for 20 min, resuspended in PBS pH 7.4, and then disrupted by ultrasonication. The supernatant was purified with glutathione agarose 4B  in PBS pH 7.4. The bound proteins were eluted with a linear gradient 10 mM glutathione in 50 mM Tris (pH 8.0). All proteins were concentrated with a Centricon apparatus . Proteins were quantitated with the Bradford assay, then filtered and stored at −70°C until use.Human SHP-2 and CSK fusion constructs were produced by consecutively subcloning the protein coding regions of SHP-2 and CSK into the pGEX-6P-1 vector . Using these constructs, human SHP-2 and CSK were overexpressed in Conjugation of 1–50 mer caveolin-1 peptide  to carboxyl beads  using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) t test to compare between sample groups, and ANOVA was used to determine differences among multiple groups. Statistical significance of the data was set at P<0.05.Statistical analyses were performed using the Student’s 2O2 induces the phosphorylation of caveolin-1 at Tyr 14 in bovine pulmonary artery endothelial cells and placenta artery endothelial cells 2O2 induces the phosphorylation of caveolin-1 in brain astrocytes, rat primary astrocytes, human astroglioma cells  and HUVECs were treated with 1 mM H2O2 for 10 min and examined by immunoblotting for p-caveolin-1. H2O2 induced the phosphorylation of caveolin-1 in the presence of H2O2 in primary astrocytes, astroglioma cells, and endothelial cells , treated with or without H2O2, and then co-immunoprecipitated with either anti-CSK or anti-caveolin-1 antibodies. An interaction between CSK and WT caveolin-1, but not Y14A mutant caveolin-1, was observed in the presence of H2O2  were phosphorylated  as described in Materials and Methods. Real-time association kinetics of either recombinant GST-SHP-2 (rGST-SHP-2) or rGST-CSK immobilized on a G1 chip with a synthetic peptide phosphorylated at a Tyr residue corresponding to Tyr 14 of caveolin-1  were analyzed by increasing peptide concentrations from 10 to 1,000 µg/mL. As shown in 2O2. In the presence of CSK siRNA, the association between caveolin-1 and SHP-2 was dramatically increased following H2O2 treatment compared to that of the control group , anti-p-Src Y27 (Y530 in human), anti-CSK, anti-SHP-2, anti-caveolin-1, anti-p-caveolin-1 and anti-tubulin antibodies. In the presence of CSK siRNA, the phosphorylation level of Src Y530 decreased following H2O2 treatment . In contrast, in the presence of SHP-2 siRNA, the level of p-Src Y530 activity upon H2O2 treatment was comparable to that of the control . To examine the effect of siRNA-mediated depletion of CSK or SHP-2 on Src-downstream signaling in response to H2O2, the same lysates were analyzed by immunoblotting against p-ERK. In the presence of CSK siRNA, the phosphorylation level of ERK was increased by H2O2 treatment .CSK is known to directly bind to caveolin-1 and suppress c-Src kinase activity by inducing c-Src phosphorylation at Try 530 2O2 for 10 min, and analyzed by immunoblotting using antibodies against p-SHP-2, SHP-2, p-Src, and Src. PTPi IV significantly reduced SHP-2 phosphorylation at Y542 and Y580 , at various concentrations (0–10 µM) for 30 min, treated with or without 5 mM H2O2-mediated oxidative stress in brain astrocytes. Our results provide initial evidence that H2O2 triggers complex formation between caveolin-1 and SHP-2, which interferes with the association between caveolin-1 and CSK and leads to enhanced activation of Src, at least in brain astrocytes.In this study, we have investigated the molecular mechanism for the positive role of SHP-2 in regulating Src activity in response to H2O2-induced Src phosphorylation was affected by SHP-2 and CSK in an inverse manner (2O2 induces phosphorylation of caveolin-1 at Tyr 14, which provides a docking site to cytoplasmic proteins, such as CSK and SHP-2. When CSK interacts with phosphorylated caveolin-1, CSK induces phosphorylation of Src Y530 to inactivate the catalytic activity of Src. However, if SHP-2 binds to phospho-caveolin-1, SHP-2 interferes with complex formation between caveolin-1 and CSK, leads to a decrease in phosphorylation of Src Y530 and an increase in phosphorylation of Src Y419, and results in sustained Src activation. These results indicate that SHP-2 and CSK reciprocally regulate H2O2-induced Src activity through competitive binding to caveolin-1 in brain astrocytes.Because both SHP-2 and CSK interacted with phospho-Tyr 14 of caveolin-1  and 5 ane manner , we prope manner . H2O2 in2O2-mediated Src signaling rather than a tyrosine phosphatase. Since SHP-2 is not a kinase, phosphorylation of Src Y419 may result from abrogating CSK action rather than inducing direct phosphorylation by SHP-2. SHP-2 is known to be either a positive or negative regulator in cellular signal transduction pathways by functioning as an adaptor protein 2O2.We observed that SHP-2 and CSK showed similar binding affinities to p-caveolin-1  and comp2O2, suggesting that caveolin-1 may function as either positive or negative regulator in Src/ERK signaling pathways depending on the kind of binding partners. In retinal ganglion cells, caveolin-1 binds to SHP-2 enabling SHP-2-TrkB interaction, leading to TrkB receptor deactivation Caveolin-1 is a major multifunctional scaffolding protein of caveolae that provides a docking site to anchor various proteins, regulates a variety of signaling molecules, and modulates downstream signaling pathways negatively or positively 2O2 strongly induced phosphorylation of caveolin-1 at Tyr 14 (2O2-induced caveolin-1 phosphorylation; however, the effect of NAC was modest compared to catalase. NAC is considered as a nonspecific ROS scavenger due to its reducing property and free radical scavenging property through increasing intracellular level of glutathione 2O2-induced caveolin-1 phosphorylation, thereby suggesting that the oxidative stress mediated by exogenously treated, in other hands, externally generated H2O2 directly induced caveolin-1 phosphorylation in astrocytes. Tyrosine phosphorylation at the 14th residue of caveolin-1 plays an essential role in providing a docking site to anchor various proteins, e.g., RhoA, E-cadherin, G-proteins, and CSK 2O2. Complex formation between SHP-2 and caveolin-1 disturbs both the binding of CSK to caveolin-1 and the negative regulation of Src by CSK. A previous report showed that SHP-2 regulates c-Src activation by controlling CSK/VE-cadherin dynamic interactions in response to vascular endothelial growth factor In both rat primary astrocytes and human astroglioma cells, Ht Tyr 14 , which i2O2-induced Src activation leads to phosphorylation of ERK 1/2 and FAK, and this phosphorylation was reduced in astrocytes by PTPi IV and SHP-2 siRNA (2O2-induced signal transduction. Since Src activation was shown to be involved in the activation of FAK in different types of cells, which plays an important role in focal adhesion and cell motility 2O2 treatment (Interestingly, our results showed that H-2 siRNA . These rreatment , which i2O2 in astrocytes. Our results suggest that SHP-2 may be one of candidate targets for oxidative brain damage, as a prominent regulator of Src signaling against excessive ROS.In summary, this study showed that SHP-2 bound to caveolin-1 and contributed to the regulation of Src activity via the competitive inhibition of CSK in the presence of H


Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear.2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar (Populus simonii × P. nigra). There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues.We used mass spectrometry combined with TiOThis study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants. Dormancy is a key feature of perennial plants. During dormancy the meristem becomes insensitive to growth-promoting signals for a period of time, before it is released and growth resumes ,2. Bud din vivo [2) microcolumn is an effective method to selectively enrich phosphopeptides [Arabidopsis [The identification of phosphorylation sites within a certain protein cannot provide a comprehensive view of the regulatory role of protein phosphorylation -17. Instin vivo ,18,23. Speptides -28. Therbidopsis ,29-40, obidopsis , rice [4bidopsis , barley bidopsis , and maibidopsis . To datebidopsis .in vivo phosphorylation sites can provide important information about the activity of protein kinases in their cellular context.Numerous cellular signaling pathways are based on the sequential phosphorylation of an array of proteins ,33,46. TPopulus simonii × P. nigra) using a MS method combined with a TiO2 phosphopeptide enrichment strategy. We identified 161 phosphorylation sites in 161 phosphopeptides from 151 proteins, most of which are associated with binding and catalytic activity. The information gained from this study provides a wealth of resources and novel insights to decode the complicated mechanisms of phosphorylation modifications in poplar. As far as we know, this is the first phosphoproteomic analysis of woody plants.To better understand the regulation mechanism of phosphoproteins and cellular signaling networks during dormancy, we investigated the phosphoproteome of dormant terminal buds (DTBs) of hybrid poplar  and rice (2.9%) [Medicago truncatula (1.3%) [Among these phosphorylation sites, 81.3% (131) of phosphorylation events occurred on Ser and 17.4% (28) on Thr (Table uncatula . Only 1.e (2.9%) ,50, but a (1.3%) . The resa (1.3%) . The speArabidopsis (80.9%) [M. truncatula (66.4%) [Arabidopsis and M. truncatula, 19.1 and 27.1% of phosphopeptides, respectively, were doubly phosphorylated [The majority (93.8%) of the 161 phosphopeptides were phosphorylated at a single residue. This value is higher than that reported for  (80.9%)  and M. t (66.4%) . Only 6.orylated ,49 , more than 75% (110) were phosphoproteins, and almost half of them were phosphorylated at equivalent site(s) or neighboring site(s) in poplar and Arabidopsis (Table Arabidopsis ortholog(s), and the remaining 65 were novel phosphorylation sites in poplar  were phosphorylated at their well-conserved C-terminal domain , Lhcb1.2 (652073) and Lhcb1.3 (715463). In the present study, we identified two previously unknown phosphorylation sites at the N-terminus; Thr38, which is well conserved across the Lhcb1 isoforms of several plants, and Thr39, which is not conserved across Lhcb1 isoforms of other plants, but is present as a non-phosphorylated residue in the Lhcb1 isoforms of Arabidopsis and spinach  in is Table ; Table 3is Table . Of the n Figure . In Popuh Figure .Medicago, rice, and Arabidopsis phosphoproteomes suggested that the phosphoproteomes are similarly conserved among various herbaceous plant species, and that overlaps are not specifically dependent on experimental conditions [Arabidopsis phosphoproteomes, providing additional evidence that phosphoproteomes overlap across plant kingdoms.Recently, overlaps among nditions . In thisArabidopsis or rice. In our study, several poplar phosphoproteins were highly conserved with their Arabidopsis ortholog(s), but their corresponding phosphorylation sites were not conserved , Medicago truncatula (MtPBA1), and rice (OsPBA1). In PtrPBA1 (673509 and 819127), there is a C-terminal motif that includes a pSer residue at position 231. This motif is conserved across two other PtrPBA1 isoforms , M. truncatula (MtG6PD), and rice (OsG6PD). However, PtrG6PD (736146) is phosphorylated at the N-terminus at residue Thr25 . Kinases with this motif regulate the activities of the vacuolar potassium channel KCO1 and the vacuolar ATPase [Confirmed phosphorylation sites are footprints of kinase activities. To date, several kinases have been documented in proteins ,33. Howeproteins . Four sir ATPase   is the major plant hormone involved in growth, dormancy, and cold acclimation . The ABAdormancy ,63,68-72dormancy ,70,73,74Arabidopsis, the DAAR protein (AT1G28330.1). Interestingly, the Arabidopsis DAAR protein is phosphorylated at its conserved Thr28 and Thr29 residues [The auxin-sensitive Dormancy-associated/auxin-repressed (DAAR) gene is associated with bud dormancy ,75,76. Iin AT1G280.1. InteArabidopsis homolog (AT5G61150.2) is also known to be phosphorylated [Arabidopsis ML (AT1G29400.2)  interacts with the FLOWERING LOCUS C-LIKE MADS-BOX PROTEIN (FLC) to activate FLC, leading to inhibition of flower development -79. Theyg AT5G611.2 is alsorylated , were prorylated . In thisg AT5G611.2 is alsn type of DHNs, one (571250) belongs to the KnS type of DHNs, and the other (818850) belongs to the SKn type of DHNs  are Group II D-11 family), late embryogenesis abundant (LEA) proteins that accumulate in response to water deficit induced by drought, low temperature, or salinity -84. Cert1 family,s, one 5750 belongbidopsis ,40. HereArabidopsis (AT5G16390.1). Interestingly, we also found another phosphorylation event related to flavonoid biosynthesis; polyphenol oxidase (PPO) (275859) was phosphorylated at Ser452  in Antirrhinum majus, a flavonoid synthase enzyme that catalyzes the formation of aurones from chalcones [Many genes related to flavonoid biosynthesis are significantly regulated during the release of dormancy, such as acetyl-CoA carboxylase (ACCase), chalcone synthase, chalcone isomerase, and flavonol synthase ,65-67. Ais AT5G1690.1. Inthalcones . To our Arabidopsis, the well-conserved Thr residue is phosphorylated in response to stress [Arabidopsis, and Medicago [The plasma membrane H+-ATPase (AHA) is responsible for the transport of protons out of the cell through the membrane . The AHAo stress ,42,97. Ho stress ,100. Geno stress ,12,66, sMedicago ,50.Some genes and proteins involved in protein biosynthesis play a role in the mechanism of bud dormancy release ,60,101. Arabidopsis [There are increases in expressions of some genes involved in energy pathways during bud release, including glyceraldehyde-3-phosphate dehydrogenase (GAPC) and phosphoenolpyruvate carboxylase (PEPC) ,12,60,93bidopsis ,110 and bidopsis . Here, wIn summary, this information on phosphoproteins in dormant poplar provides a useful dataset, and provides new insights for exploring the relevance of phosphorylation for dormancy. However, further research, e.g., comparing proteomes between dormant/non-dormant tissues, is required to clarify the roles of phosphorylation in the dormancy process.Arabidopsis or in rice. Therefore, it is important to determine phosphorylation sites in poplar proteins, and to determine the roles of these phosphorylations in modifying protein function during growth and development. To date, there have been no extensive studies on the poplar phosphoproteome. In this work, we conducted a detailed analysis of the phosphoproteome of dormant poplar buds using an MS method and TiO2 phosphopeptide-enrichment strategies. We found 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins, most of which are associated with binding and catalytic activity. Most of the poplar phosphoproteins have orthologs in Arabidopsis, suggesting that there are similar signaling pathways mediated by phosphorylation in poplar and Arabidopsis. However, some phosphoproteins and phosphorylated sites were unique to poplar, thus confirming the need to obtain phosphoproteome data from poplar. Several phosphorylation motifs were extracted from the dataset by Motif-X. This could provide evidence for the involvement of kinases in phosphorylation of these identified proteins during dormancy in poplar. Further experiments are now required to confirm that these specific kinases interact with the identified phosphoproteins in vivo. A promising way forward is to comprehensively characterize and analyze the dynamics of phosphorylation of poplar proteins in response to environmental changes, using specialized targeted quantitative proteomics tools.Many physiological features of woody plants are not reflected in the herbaceous model Populus simonii × P. nigra) in Harbin, China,  at the end of December, 2009. Samples were frozen in liquid nitrogen and stored at -80°C until use.Dormant terminal buds were collected from hybrid poplar (Iodoacetamide (IAA) and dithiothreitol (DTT) were purchased from Acros Organics . HPLC-grade acetonitrile (ACN) was obtained from JT Baker . HPLC-grade water was prepared using a Milli-Q A10 system from Millipore . ModiWed sequencing-grade trypsin was supplied by Promega . Protease-inhibitor cocktail and the 2-D Quant kit were obtained from Amersham Pharmacia Biotech . All other reagents were purchased from Sigma .g at 4°C for 1 h, and the precipitates were washed with cold acetone containing 0.07% (v/v) 2-mercaptoethanol. The pellets were dried by vacuum centrifugation and dissolved in 7 M urea, 2 M thiourea, 20 mM dithiothreitol, 1% (v/v) protease-inhibitor cocktail, 0.2 mM Na2VO3, and 1 mM NaF at room temperature for 2 h, before centrifugation at 40000 g at 10°C for 1 h. The resulting supernatant was collected and kept at -80°C until further use. The total protein content of the samples was quantified using a 2-D Quant kit.The dormant terminal buds were crushed into a fine powder in liquid nitrogen and resuspended at -20°C in 10% (w/v) trichloroacetic acid (TCA) in cold acetone containing 0.07% (v/v) 2-mercaptoethanol for at least 2 h. The mixture was centrifuged at 10000 2 was added to a final concentration of 20 mM. Then, endoprotease Lys-C was added to a final substrate-to-enzyme ratio of 100:1, and this reaction was incubated for 12 h at 37°C. The Lys-C digest was added to 1 M urea containing 100 mM ammonium bicarbonate, and modified trypsin was added to a final substrate-to-enzyme ratio of 50:1. The trypsin digest was also incubated at 37°C for 12 h. After digestion, the peptide mixture was enriched using TiO2 microcolumns for further MS analysis.Total proteins were digested as described elsewhere ,112. Bri2 microcolumns were packed as described elsewhere [2 beads were suspended in 100% ACN, and an appropriate volume of this suspension (depending on the size of the column) was loaded into the GELoader tip. Gentle air pressure produced by a plastic syringe was applied to pack the column. The TiO2 microcolumn was equilibrated with loading buffer . Immediately, the trypsin-digested peptide mixture diluted in loading buffer was added to the TiO2 microcolumn. Then, the column was washed once with loading buffer (40 μl) and three times with washing buffer . The washing and loading buffer contained 80% ACN organic solvent in order to abrogate the adsorption of peptides to the C8 material [The TiOlsewhere . A smallmaterial . The bou18 5-μm, 180-μm × 20-mm pre-column and a BEH C18 1.7-μm, 75-μm × 250-mm analytical reversed-phase column (Waters) were used. The MassLynx  program was used for instrument control and data acquisition. The mobile phases were (A) 100% H2O/0.1% formic acid and (B) 100% ACN/0.1% formic acid. The samples were dissolved in aqueous 0.1% formic acid solution and loaded onto the pre-column at a flow rate of 5 μl/min for 3 min. The phosphopeptides were separated by a gradient of 5-40% mobile phase B for 90 min at a flow rate of 200 nl/min, followed by a 10-min rinse with 90% mobile phase B. The column was re-equilibrated with the initial conditions for 20 min. The lock mass was delivered from the auxiliary pump of the NanoAcquity pump at a constant flow rate of 400 nl/min at a concentration of 100 fmol/μl of (Glu1) fibrinopeptide B to the reference sprayer of the NanoLockSpray source from the mass spectrometer. In this study, every sample was analyzed in triplicate. Data-dependent acquisition was carried out in positive ion mode. MS spectra were acquired for 1 s from mass-to-charge ratios of (m/z) 350 to 1990. Two of the most intense precursor ions that were doubly or triply charged were selected from m/z 350 to 1990. MS/MS spectra produced by collision-induced dissociation (CID) were acquired for 2 s from m/z 50 to 1990. The collision energy was automatically calculated according to peptide charge and m/z; a dynamic exclusion window was applied to prevent the same m/z from being selected for 2 min after its acquisition. The candidate phosphopeptides were initially assigned by ESI-MS/MS using 79.96-Da mass increments per phosphate moiety relative to the unmodified peptides. To detect the phosphopeptides, we utilized the preferred loss of the phosphate group upon collision-induced dissociation. In positive ion tandem MS, an intense neutral loss of 98 Da, corresponding to H3PO4, was observed for peptides containing phosphorylated Ser, Thr, and Tyr residues.NanoUPLC-ESI-MS/MS was performed with a splitless nanoUPLC  in combination with a Synapt high-definition mass spectrometer with a nanospray ion source (Waters). A symmetric CPopulus trichocarpa v1.1 (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) protein sequence database using an in-house Mascot server (version 1.8) with acetylation in the N-terminus of the protein, carbamidomethylation, methionine oxidation, and phosphorylation of serine/threonine/tyrosine residues as variable modifications. Two missed cleavage sites were allowed. The search was performed with a peptide mass tolerance of 15 ppm in the MS and 50 ppm in the MS/MS modes. The false discovery rate (FDR) was 0.00% for peptide matches above the identity threshold and 0.36-0.85% for peptide matches above the homology or identity threshold.The MS/MS data were processed and converted to a pkl file format with ProteinLynx software (Waters), and the resulting pkl file was used to search against the JGI http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) by their protein ID. The Blast2Go program [http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default parameter settings. Protein functions, annotations, and classifications were also examined using gene ontology (GO), GO-EnzymeCode, and InterPro databases and search tools.Using a custom Perl program, all the phosphoprotein sequences were extracted from protein databases ( program  was usedhttp://pfam.sanger.ac.uk/search) was applied to obtain Pfam information for identified phosphoproteins. The significantly enriched phosphorylation motifs set was extracted from our phosphopeptide data using the Motif-X algorithm [Populus trichocarpa v1.1 in Fasta format (in a shortened version due to upload restrictions of 10 MB) were used. The occurrence threshold was set to 5% of the input data set at a minimum of three peptides, and the probability threshold was set to P <10-5. Amino acid sequences around the phosphorylated amino acid based on the alignment of all the phosphorylation sites were completed by the Weblogo program [The Batch sequence search tool . File contains all the original MS/MS spectra of 161 phosphopeptides identified in this study.Click here for fileComparison of singly and doubly phosphorylated peptides.Click here for fileLocation of phosphorylation sites in characterized conserved domains.Click here for fileArabidopsisFlowchart for analyzing the conservation of phosphoproteins and phosphosites between poplar and .Click here for fileConserved phosphorylation sites within orthologous proteins. (a) Phosphosites conserved in orthologous proteins. (b) Phosphosites that were not conserved in orthologous proteins.Click here for fileUnconserved phosphorylation sites within orthologous proteins.Click here for filePopulus trichocarpa genomeKOG analysis of identified phosphoproteins and all proteins encoded in . (a) Percentage of KOG functional group categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome. (b) Percentage of KOG functional subgroup categories from the identified phosphoproteins and all proteins encoded in Populus trichocarpa genome.Click here for filePopulus trichocarpa genomeComplete list of KOG analysis of phosphoproteins and all proteins encoded in .Click here for fileArabidopsisSequence alignment of phosphorylated sites in protein kinases between poplar and .Click here for fileDetailed information for identified phosphoproteins referred to in discussion section.Click here for fileArabidopsisSequence alignment of phosphorylated sites in protein phosphatases between poplar and .Click here for fileArabidopsisSequence alignment of RPS12 between poplar and .Click here for fileArabidopsisSequence alignment of conserved N-terminus of eIF5A between poplar and .Click here for fileArabidopsisSequence alignment of conserved C-terminus of EF-1-alpha between poplar and .Click here for file


Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.+ enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (KmVox) and phosphorylation (KmVp) rates. We also demonstrate that determination of KmVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.The proposed method is based on the simultaneous monitoring of substrate oxidation  and phosphorylation (determined using the glucose - hexokinase - glucose-6-phosphate dehydrogenase - NADPBecause the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions. In most tissues, such as skeletal muscle, mitochondria are the main site of energy production through the oxidative phosphorylation pathway. In addition to their crucial role in energy metabolism, mitochondria also take part into other important physiological functions such as calcium homeostasis, apoptosis signaling and ROS production. All these roles are in complex and close interaction  However, the identification of the functional consequences of mitochondrial dysfunctions on cellular energetics remains a scientific challenge. Indeed, most studies focus on isolated parts of mitochondrial oxidative phosphorylation, making the link between mitochondrial dysfunctions and their consequences on cellular energetics difficult to decipher. It is also well established in the field of systems biology that the emergence of unexpected properties can arise from the cooperative interactions between individual components of a system  The aim of the present paper is to propose a new and easy to use method allowing a true and accurate determination of oxidative phosphorylation affinity for ADP using isolated mitochondria. This method is based on the direct and simultaneous independent monitoring of oxidation and phosphorylation rates during true steady states under various ADP concentrations (from very low (7 µM) to saturating ADP concentrations), the actual ADP and ATP concentrations being determined by bioluminescent assays. We demonstrate how this method can provide other useful data about the energetics of mitochondria in the cell, since its application also allows the direct determination of mitochondrial coupling efficiency. To achieve this aim, mitochondria isolated from muscle and liver, two populations known to present significant differences, were studied in order to validate the method.All experiments were conducted in agreement with the National and European Research Council Guide for the care and use of laboratory animals in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”. All animal protocols used were approved by our local ethics committee named Direction départementale des services vétérinaires de la Gironde, the corresponding license number being P.D. 3308010 (11/19/2008).−1). The gastrocnemius muscle and the liver were then dissected and washed separately in the isolation medium containing 100 mM saccharose, 180 mM KCl, 50 mM Tris, 5 mM MgCl2, 10 mM EDTA and 0.1% (w/v) BSA (pH 7.2). Before homogenization, gastrocnemius muscle was minced and exposed during 5 minutes to protease . Mitochondria were extracted as described in Isolation of liver and skeletal muscle mitochondria from 6 month-old male Wistar rats were anesthetized by isofluran inhalation and killed by intraperitoneal injection of pentobarbital  BSA (pH 7.2). Glutamate (5 mM)+Malate (1 mM)+succinate (5 mM) were used as substrates in all experiments.Oxygen consumption and ATP synthesis rate were monitored simultaneously in a glass vessel  in a medium containing 240 mM Manitol, 100 mM KCl, 1 mM EGTA, 20 mM MgCl2 concentration in air-equilibrated medium was taken as 240 µM Oxidation rates were determined polarographically with a Clark electrode (Rank Brothers). O−1, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase  - NADP+ (1.6 mM) + as electron acceptor (eq. 2). ATPPhosphorylation rates were determined using a coupled enzymatic system composed of Glucose (5 mM) - Hexokinase . Mitochondrial protein concentration in the oxygraphic vessel for each recording was 25 µg.ml−1 in order to decrease mitochondrial non-specific absorption at 340 nm.Consequently, NADPH production is stoichiometrically linked to mitochondrial ATP synthesis rate, which can therefore be monitored by spectrometrically measuring the increase in the absorbance of NADPH at 340 nm. Conversion of NADPH absorbance into NADPH concentration was performed using a molar extinction coefficient of 6.22 mM To avoid any interference by residual adenylate kinase activity, an excess of P1,P5- Di(adenosine-5)pentaphosphate  was added to the measurement medium in all experiments.ADP and ATP concentrations were determined both at the onset of steady state and just before the end of each recording to assess the stability of nucleotides concentrations. The following protocol was optimized for concentrations ranging from 5 to 50 µM corresponding to the sensitivity range of the ATP monitoring reagent used  diluted 625 times:150 µl of the ATP monitoring reagent were added to a luminometer cuvette. Cuvette was then placed in the luminometer chamber  to record the emitted light by the reagent alone. This value was subtracted to the value obtained during the step 2 for accurate determination of ATP concentration.10 µl of the measured medium were taken in the oxygraphic vessel using a 10 µl Hamilton syringe and instantaneously added to the cuvette prepared during step 1. The amount of emitted light gave ATP concentration in the sample.−1 final) were then added to the luminometer cuvette in order to convert all ADP into ATP. Subtracting the ATP concentration determined during step 2 allowed the determination of the ADP concentration in the assay.Phosphoenol pyruvate  and pyruvate kinase  and phosphorylation (KmVp) rates. KmVox and KmVp for ADP were determined by fitting respectively oxidation and phosphorylation rates expressed as a function of the ADP concentration by the Michaelis-Menten equation (eq. 3):m corresponds to the Michaelis-Menten constant.All experimental data were treated with a specific homemade program developed using the Igor Pro software (Wavemetrics). As both oxidation and phosphorylation rates were simultaneously determined for each ADP concentration, mitochondrial apparent Km for ADP could be determined for both oxidation . To face this challenge, we combined and adapted existing methods in an original way to investigate mitochondrial bioenergetics and its regulation by ADP. To demonstrate the discriminating capabilities of the proposed method, we chose to compare mitochondria isolated from skeletal muscle and liver . Indeed, it has been shown that mitochondrial oxidative phosphorylation significantly differs in these two tissues The complete study of mitochondrial oxidative phosphorylation cannot be restricted to respiratory chain activity. Indeed, due to proton leak dependency on membrane potential (Δψ), the degree of coupling between oxidation and phosphorylation is known to largely vary depending on the rate of ATP synthesis and Δψ + in order to determine the phosphorylation rate, since NADPH produced is stoichiometrically linked to mitochondrial ATP production The glucose-hexokinase enzymatic system is widely used as ADP-regenerating system m for ADP, a parameter that is related to the mitochondrial capacity to respond to changes in ATP demand and primarily reflects the kinetic properties of both the adenine nucleotide translocator and the F0-F1 ATP-synthase.Mitochondrial affinity for ADP can be investigated by determining the apparent KmVox and KmVp. As shown in mVox and KmVp as compared to liver mitochondria, clearly indicating a much higher affinity for ADP in muscle mitochondria.In the present study, both oxidation and phosphorylation rates were therefore determined as described above during steady states under measured ADP concentrations ranging from 7 to 900 µM. The dependence of oxidation and phosphorylation rates on ADP concentration for liver and muscle mitochondria are shown in mVp was significantly higher as compared to KmVox in both tissues . This can easily and accurately be done in isolated mitochondria using the proposed method.Interestingly, the K tissues . During mVp to KmVox ratio was significantly higher in liver mitochondria . Taken all together, these results emphasize the interest of the determination of both KmVox and KmVp, since the information provided by each of these parameters are complementary in order to characterize the integrated functioning of oxidative phosphorylation. In addition, major functional differences in the phosphorylation rate response to an increase in the ADP concentration were revealed under low ADP concentrations , this method could therefore be applied to study mitochondrial bioenergetics in small amount of tissue from knock-out mice models or in human biopsies.When using the method presented here, phosphorylation rates are directly determined during true steady states, making consequently the determination of oxidative phosphorylation affinity for ADP accurate. The concomitant determination of the corresponding oxidation rates also brings crucial information since P/O ratio can be calculated. As a consequence, any modification of the inner mitochondrial membrane properties (i.e. modification of proton leak) may be revealed after application of the present method. In addition, the direct determination of both ADP and ATP concentrations increases the precision in the determination of mitochondrial affinity for ADP. This method consequently represents an easy way to obtain a complete and accurate check-up of integrated mitochondrial bioenergetics with a special emphasis on low ADP concentrations, likely to occur under Our method could therefore be of some importance to determine whether mutations or alterations demonstrated at the molecular level can lead or not to true functional alterations. This method therefore opens the route to a better understanding of functional consequences of mitochondrial dysfunctions arising in pathologies.