Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward “on/off” response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. CyanobacteriaStreptococcus pneumoniae phosphorylates the orphan response regulator RitR StreptococcusBacillus subtilis, identified the two-component system histidine kinase DegS as being phosphorylated on the residue serine76, located in the signal sensing domain Two-component systems are a ubiquitous means of signal transduction in bacteria B. subtilis strain demonstrated that DegS/U also affects motility, complex colony and biofilm formation. The regulation was shown to depend on the discrete levels of DegU phosphorylation, in a manner far more subtle than a simple on/off switch degSU transcription or modulate the activity of synthesized DegS/U proteins. The degSU genes are transcribed as an operon and degU is itself transcribed from two additional promoters: one activated by DegU∼P and the other by nitrogen starvation degR expression is SigD dependent and peaks in late exponential phase, but the physiological role remains elusive Early mutational studies of the DegS/U two component system established that the response regulator binds DNA sequences and regulates expression of specific genes both in its phosphorylated and unphosphorylated state. This was exemplified by reciprocal effects of the two forms of DegU on exoprotease production and competence in vitro, and a phosphomimetic mutant of DegS leads to an increased DegU∼P pool, and influences the transcription of key DegU-dependent promoters in vivo.Despite the fact that the DegS/U two-component system is submitted to an elaborate control at both transcriptional and protein level, no specific DegS-activating signal has so far been proposed. Most two-component system histidine kinases are transmembrane proteins, presumably activated by extracellular signals via their N-terminal signal-sensing domains protruding from the cell surface in vitro phosphorylation experiments with purified DegS and 32P-γ-ATP. Phospho-histidine and phospho-serine can be easily distinguished, the latter being stable in acidic conditions and resistant to heat. Since the entire radioactive label present on autophosphorylated DegS was removed by acid and heat treatment  and PrkC were unable to phosphorylate DegS, whereas both YbdM and YabT tested positive for DegS phosphorylation  was about 50% more competent than the wild type, while the degS S76D mutant exhibited an approximately 5-fold reduction in competence (18+/−0%), concurring with our working hypothesis. Next, competence development on single cell level was assayed by introducing GFP under control of the comK promoter. In this system we further assayed the effects of inactivating individual serine/threonine kinase-encoding genes prkC, ybdM and yabT .After confirming the effect of the phosphomimetic mutation S76D on the pool of DegU∼P using the B medium . A signiyvcA promoter activity prompted us to investigate the effects of the degS S76D mutation on this and the other social behavioural traits pellicle formation and swarming that are regulated by low levels of DegU∼P. The laboratory strain B. subtilis 168 does not readily swarm due to defects in surfactin production and a frame shift mutation in swrAdegS S76A colonies were capable of producing larger and more complex aerial structures than the strain degS S76D  of about 60 min, it started swarming at the same speed as the wild type until reaching about one half-distance towards the end of the plate. Then it paused for about 120 min, seemingly consolidating. Finally, it continued swarming and went on to reach the end of the plate with about 180 min delay compared to the wild type. Due to this stop-and-go behaviour, the degS S76D final swarm exhibited concentric layers at various stages of consolidation (degS S76D. It has previously been reported that expression of the flagella operon is inhibited at high DegU∼P levels degS S76D consolidation (data not shown). Further, this phenotype does not seem to be related to either surfactin production or the swrA mutation since a lab strain cured for these mutations, DS155 degS S76D mutation indeed leads to an increased DegU∼P level. If this mutation can mimic the phosphorylated form of DegS in vivo, it would indicate that the serine phosphorylation event could have the potential to regulate social behaviours regulated at low DegU∼P levels in B. subtilis.A more apparent phenotype was observed in swarming . The wil 300 min . The deglidation . It is din vitro. YbdM-dependent phosphorylation was specific for serine 76 and it led to increased efficiency of phosphotransfer to DegU. Moreover, ybdM overexpression led to a similar  in vivo effect on some DegU-controlled promoters as the degS S76D phosphomimetic mutation. The degS S76D strain exhibited phenotypes corresponding to elevated levels of DegU∼P in vivo. The use of point-mutations to mimic phosphorylated and non-phosphorylatable proteins is a common tool employed in protein phosphorylation research but it has a potential pitfall. The ensuing phenotypes may be caused by conformational changes caused by the mutations, which are entirely unrelated to phosphorylation. In case of the DegS S76D mutation, the protein behaves in a similar manner as wild type DegS phosphorylated by YbdM. The fact that the mutant protein exhibits a higher DegU kinase activity is expected since it would correspond to 100% phosphorylation of DegS, which is never achieved by incubating DegS with YbdM in vitro. The non-phosphorylatable mutant DegS S76A also exhibits an increased DegU kinase activity in vitro but this does not translate to an increased pool of DegU∼P in vivo. Whether this could reflect a conformational change of the protein leading to increased kinase activity or maybe increased stability in vitro remains elusive. Knocking out the kinase YbdM did not provoke strong phenotypes, possibly due to complementation of its function by remaining Hanks kinases. The overproduction of the regulatory kinase YbdM, which possibly phosphorylates other proteins, could also lead to non-physiologically relevant phenotypes. The fact remains that there is an agreement between the observations with the phosphomimetic DegS S76D and overexpression of YbdM. If DegS S76D indeed can mimick the serine 76-phosphorylated state of DegS, it would suggest that phosphorylation of serine 76 of DegS contributes to an already very complex process of regulating the level of DegU phosphorylation in B. subtilis. The molecular mechanism by which serine phosphorylation activates DegS activity remains elusive. The fact that a phosphomimetic mutant DegS S76D behaved in a similar manner would preclude that phospho-transfer from serine to either DegS histidine or DegU aspartate is involved. It would seem plausible that phosphorylation could induce a conformational change thereby stimulating kinase activity, but due to the lack of any structural data for DegS this is merely a speculation. The residue serine 76 of DegS was found to be phosphorylated in the late exponential growth phase B. subtilis can possibly contribute to phosphorylating DegS serine 76 in vivo to some extent. These kinases are presently almost completely uncharacterised (except PrkC) and more work will be needed to elucidate specific signals that trigger their expression, activity and substrate specificity. Our results indicate that DegS serine phosphorylation influences DegU phosphorylation in vivo, pointing towards a possible role in regulating phenomena such as motility and complex colony formation.Here we describe, to the best of our knowledge, the first example of a bacterial two component sensory kinase that is regulated via serine phosphorylation of its input domain by a Hanks type Ser/Thr kinase. DegS was phosphorylated by Hanks type serine/threonine kinases YbdM and YabT E. coli NM522 was used for plasmid propagation in cloning experiments. The chaperone overproducing strain E. coli M15 carrying pREP4-GroESL B. subtilis strains used in this study are listed in E. coli and B. subtilis cells were grown at 37°C with shaking in LB medium. In addition, B. subtilis was grown in competence media for transformation experiments as described E. coli ampicillin (100 µg/mL), kanamycin (25 µg/mL), tetracycline (8 µg/mL) and for B. subtilis erythromycin (5 µg/mL), neomycin (5 µg/mL) and tetracycline (15 µg/mL) were added as appropriate.B. subtilis genes degS, degU, prkA, prkC , yabT  and ybdM were PCR-amplified using genomic DNA from the strain 168 as template degS S76A and S76D were obtained using two partially overlapping mutagenic primers  was constructed using two partially overlapping mutagenic primers. The PCR product and pHT315 EcoRV and PvuII and the fragments ligated. The resulting vector was devoid of the Gram-positive origin of replication. Upon transformation and integration on the chromosome, the mutation was verified by sequencing. Inactivation of prkA, prkC, ybdM and yabT was done using pMUTIN2 ybdM was inserted in pHT315 and introduced in B. subtilis strains bearing promoter-lacZ fusions. The vector pG+host8 containing a temperature sensitive origin of replication was used to introduce degS S76A and S76D point mutations in situ, replacing the chromosomal version of degS in B. subtilis 168 BamHI-Cfr9I fragments from pQE30-degS S76A and S76D containing the mutated gene were inserted into pG+host8 between the BamHI and Cfr9I sites. B. subtilis was transformed with the constructs, plated on tetracycline-containing plates and incubated at the non-replicative temperature 37°C, which selects for integration of the vector on the chromosome by single crossing-over. The transformants were further cultured in liquid LB for loss of plasmid by the second crossing-over event and the chromosomal mutation was verified by sequencing. In our hands the second-crossing over happened with a low frequency indicating that B. subtilis, contrary to Lactococcus lactis, was not severely affected by a chromosomal copy of pG+host8 actively replicating. Point mutations in B. subtilis NCIB3610 were introduced by same method except that transformation with pG+Host8 was done by PEG treatment of protoplasts  primers . The PCRE. coli M15 carrying pREP4-groESL. Cultures were grown shaking at 37°C to OD600 0.5, induced with 1 mM IPTG and grown for an additional 3 hours. Cells were disrupted by sonication and 6xHis-tagged proteins were purified on Ni-NTA columns (Qiagen) according to manufacturer's instruction, desalted with PD-10 columns  and stored in a buffer containing 50 mM Tris-Cl pH 7.5, 100 mM NaCl and 10% glycerol. Protein concentrations were estimated using the Bradford assay (Bio-Rad) with BSA as standard.6xHis-tagged proteins were synthesised in the chaperone-overproducing strain 32P-γ-ATP (20 µCi/mmol), 42.5 mM Tris-Cl (pH 7.5), 5 mM MgCl2, 85 mM NaCl and 8.5% glycerol. For PrkC, we varied the pH value of the Tris-Cl buffer to the additional pH values of 7.0, and 8.0. To measure the influence of YbdM on DegS phosphotransfer to DegU, DegS was pre-incubated with YbdM for 3 h, in exactly the same conditions as described above, only with non-labelled ATP. Reactions were started by addition of ATP, incubated at 37°C for 60 min (unless otherwise indicated in figure legends) and stopped by addition of SDS-containing loading buffer. All gels shown in the same figure have the same exposure times. For dephosphorylation assays, after the initial phosphorylation reaction described above, the DegS/YbdM and DegS/YabT reaction mixtures were desalted on a PD-10 column (to remove the ATP), lyophilized and resuspended in the identical reaction mixture as before, but without ATP, and incubated 2 hours at 37°C. The proteins were separated by SDS-PAGE (for separation of PrkC and DegS we used a Tris-tricine gel). For detection of phospho-histidine and phospho-aspartate, the gels were rinsed with water and dried directly, whereas for detection of phospho-serine they were additionally treated in boiling 0.5 M HCl for 10 min. Radioactive signals were visualised using STORM phosphoimager and quantified using the ImageQuant software .Phosphorylation reactions were performed in a total volume of 30 µl, essentially as described 600 of 0.02 and grown with shaking at 37°C. IPTG at a final concentration of 0.5 mM was added where appropriate. At time points indicated in the figures, 2 mL samples were taken, spun down  and cell pellets were stored at −20°C. The pellet was resuspended in 2 mL of Z-buffer  and OD600 was measured. 1 mL cell suspension was treated with 0.5 mg lysozyme for 5 min at 30°C before adding 8 µL 10% Triton X-100 and incubating for additional 5 min. Reaction was started by addition of 100 µL of 4 mg/mL ONPG and stopped by addition of 1 mL of 0.5 M Na2CO3. Miller units were calculated as described previously 150 mL LB was inoculated with overnight culture to ODB. subtilis strains cells were transformed according to the two-step protocol described by Yasbin et al. with the modification that 60 min after dilution in GM2 medium, 0.5 mL culture were transferred to three test tubes containing 1 µg DNA and incubated an additional 90 min before plating. The plasmid pDG268-neo To assess the competence of 600 0.5 at which point 5 µL culture was spotted on a dried MSgg plate (1.5% agar) and incubated for 96 hours at 28°C. Colonies were measured and photographed using a SONY Cyber-shot DSC-T20 camera with close focus enabled. For each sample, a representative image from 20 examined colonies is presented.Cells were grown in LB shaking at 37°C to OD600 of 0.5 at which time LB plates (0.7% agar) dried for 20 min in a fume hood (face up) where inoculated with 5 µL culture and dried an additional 10 minutes. Petri dishes were sealed with parafilm to avoid plates drying out and incubated at 37°C. Swarm radii were measured at the times indicated in the figure. Plates were scanned about 1 hour after the swarm reached the edge of the plate using a standard HP office scanner.Cells were grown in LB to an OD


The abnormal BCR-ABL oncoprotein is a constitutively active tyrosine kinase driving aberrant proliferation of transformed hematopoietic cells. BCR-ABL regulates activation of many mitogenic and pro-survival pathways, including the PI 3'K/AKT/mTOR pathway that controls various effectors and regulates initiation of mRNA translation in mammalian cells. Although tyrosine kinase inhibitors (TKIs) that target the ABL kinase domain have remarkable clinical activity and have dramatically changed the natural history of Ph+ leukemias, resistance to these agents also develops via a wide range of mechanisms. Efforts to target the PI3'K/AKT/mTOR signaling pathway using kinase inhibitors have been the focus of extensive ongoing investigations by several research groups. Here we review the effects of activation of the AMPK kinase, which regulates downstream targeting and inhibition of mTOR. The potential for future clinical-translational applications of AMPK activators such as AICAR, metformin and resveratrol for the treatment of chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are discussed. Bcr-Abl fusion gene is the oncoprotein BCR-ABL, which is expressed in CML and Ph+ ALL .2 q34;q11 Ph+ ALL .The identification of imatinib mesylate  as a small molecule ATP-pocket inhibitor of BCR-ABL dramatically re-defined the treatment of CML and had a major impact in the survival of patients with CML and Ph+ ALL -13. As tDespite such advances in the field, mutations rendering CML and Ph+ ALL patients non-responsive to TKI's have been identified, including the threonine 315 to isoleucine (T315I) mutation and several others, which differentially prevent binding of different TKIs to the active site of the ABL kinase domain, thereby evading inhibition . Beyond Kip1 [in vitro [Among the multiple cellular cascades that are activated by BCR-ABL, the PI3'K/AKT/mTOR pathway , 28-30 iKip1  and decrKip1 . Furtherin vitro .Downstream of the PI3'K/AKT pathway, the mTOR signaling cascade is also hyperactive in CML , 36-38. Beyond agents that directly target and inhibit TORCs, indirect suppression of mTOR function by modulation of the AMP-activated Protein Kinase (AMPK) pathway may provide an important alternative therapeutic approach . AMPK reIn one of the initial studies in which the effects of modulation of AMPK in ALL cells were assessed, the AMP-analog AMPK-activator compound 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) was shown to exert antiproliferative effects on childhood ALL cells, including the SupB15 cell line which expresses the p185 BCR-ABL fusion protein . In thatResveratrol, a naturally occurring substance found in grapes, has been shown to modulate AMPK in BCR-ABL transformed cells and to exhibit antileukemic effects -58. TreaIn a recent study , we asseThere have been remarkable advances in the treatment of CML over the last decade. With the introduction of first and second generation TKIs in the treatment of CML, the majority of patients achieve complete hematological, cytogenetic and molecular responses , 12. Als


Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming.inact/KI of 36.7±13.6 M−1s−1 using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with kinact/KI values of 406.4±10.8 and 36.5±1.3 M−1s−1. No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS) by 4-aminophenols in various redox environments was determined by the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H2O2 is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid.In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a kIn this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases. Cathepsins are lysosomal cysteine proteases belonging to the papain-subfamily C1A of the clan CA of cysteine proteases Much is now known about the cysteine cathepsins. Their crystal structures, physiological and pathological involvement as well as their regulatory mechanisms constitute them as attractive targets for drug discovery 2O2 generated by such molecules induces oxidation of accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, in this way causing false positive inhibition of several classes of proteases, such as protein tyrosine phosphatases In the last decade a new mechanism of false positive inhibition has been reported for proteases that possess functional groups susceptible to redox modification 1), displays time-dependent inhibition. The inhibition has been shown to be concentration dependent, nonspecific, irreversible and active site directed, using enzyme kinetics, mass spectrometry, the DCG-04 probe and isoelectric focusing (IEF). Furthermore, the inhibition is shown to depend on reactive oxygen species (ROS) using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and is abolished by catalase. Here we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in screening assays for inhibition of cysteine proteases.We identified 5-nitroquinolin-8-ol as a selective and potent inhibitor of cathepsin B using virtual-screening, enzyme kinetics and cell-based assays in a previous study 1) (95%), 5-nitroquinolin-8-ol (2) (96%), 4-aminophenol (6) (≥99%), phenol (7) (∼ 99%), and aniline (8) (≥99.5%) were purchased from Sigma-Aldrich and used without further purification. 4-Aminonaphthalen-1-ol hydrochloride (3) (90%), quinolin-8-ol (4)  and quinolin-5-amine (5)  were purchased from Acros Organics and used without further purification (2O2 from Merck. Irreversible cathepsin B specific inhibitor CA-074 was obtained from Bachem and the active site probe DCG-04 was kindly provided by Dr. Matthew Bogyo.5-Aminoquinolin-8-ol (fication . CystineE. coliHuman recombinant cathepsin B was prepared as reported st + (vi − vs)(1 − e−kobst)/kobsP  =  v, where P denotes the product concentration in relative fluorescence units (RFU) at time t; iv and sv are the initial and steady-state velocities and obsk is the apparent first-order rate constant for the establishment of the final steady-state equilibrium. Plots of obsk values versus inhibitor concentration yielded a linear fit describing a single-step mechanism for the irreversible inactivation of the enzyme, with a slope corresponding to the second-order rate constant inact/KIk.To evaluate the inhibitory activity of the tested compounds the following substrates were used: Z-Arg-Arg-AMC (Bachem) at 60 µM, Z-Phe-Arg-AMC (Bachem) at 2 µM and Arg-AMC (Biomol) at 10 µM for cathepsins B, L and H, respectively. 5 µl of inhibitor or dimethyl sulfoxide (DMSO) and 5 µl of the appropriate substrate were added to the wells of a black 96-well microplate. The reaction was initiated by adding 90 µl of cathepsins B, L or H at final concentrations of 400 pM, 200 pM or 2.5 nM, respectively. Formation of fluorescent degradation product was monitored continuously at 380 nm±20 nm excitation and 460 nm±10 nm emission on a Tecan Safire2 reader (Tecan) using minimal kinetic interval at 37°C. All kinetic measurements were performed in duplicate. Progress curves obtained with a range of concentrations of inhibitor were fitted to the equation 2DCFDA  was used as described elsewhere with minor modifications 2DCFDA was dissolved in DMSO at 1 mM, diluted to 100 µM with 0.01 N NaOH and kept in the dark at room temperature (RT) for 30 min. The reaction was performed in a black 96-well microplate, adding to each well 10 µL of the de-esterified probe solution, 5 µl of DMSO or inhibitor at a final concentration of 500 µM and 85 µl of the activation buffer with different redox agents . In a parallel experiment catalase from bovine liver  was added to the activation buffer to a final concentration of 200 U/ml. The reaction was continuously monitored for 15 min using 535 nm excitation and 485 nm emission filters on a Tecan GENios reader (Tecan), with 1 min kinetic interval at 37°C. All measurements were performed in duplicate.H2O2.1 µg cathepsin B in the activation buffer with a suitable redox agent at 5 mM was incubated with 200 µM respective inhibitor for 1 h at RT. Samples were labeled with 2 µM DCG-04 for 1 h at RT, separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. The latter was blocked overnight with 0.5% Tween 20 in PBS at 4°C and incubated with 1 µg/ml streptavidin-horseradish peroxidase (Sigma-Aldrich) for 1 h at RT. The membrane was washed five times and the spots on the membrane were visualized with 0.5 mg/ml 3,3′-diaminobenzidine (Sigma-Aldrich) and 0.0005% (v/v) H1 or H2O2 at 500 µM or 490 µM, respectively for 5 h at RT. 4 µl of each sample was loaded on the precast isoelectric focusing gel PhastGel IEF (pI range 3.0–9.0)  and separated on the Phast System  according to the protocol of the manufacturer. After separation, gels were developed on the Phast System using Coomassie blue staining. pIs of individual bands were determined from a calibration curve  or DMSO were added and incubated with cathepsin B for additional 5 h at RT. The mixtures were then dialyzed against water using Microcon Centrifugal Filter Devices YM-10 (Millipore). Samples were prepared by adding an equal volume of acetonitrile containing 0.1% formic acid and analyzed by ESI-MS on an AutoSpec Q instrument (Micromass).Cathepsin B (4.4 µM) was incubated with DMSO or CA-074 (44 µM) in the activation buffer containing 1 mM cysteine for 1 h at RT. Compound 1 (obsk) versus inhibitor concentration yielded a linear fit describing a single-step mechanism for irreversible inactivation of the enzyme with a second-order rate constant, inact/KIk, of 36.7±13.6 M−1s−1   . A repre1 was a cathepsin B specific inhibitor we tested the compound against two related cathepsins, the aminopeptidase cathepsin H and the endopeptidase cathepsin L. Results show that compound 1 inhibited cathepsin H in a manner similar to cathepsin B under non-reducing conditions (redox-free and 5 mM cystine) since the inactivation rates remained almost the same  , only compounds 1 and 3 retained time-dependent irreversible inactivation of cathepsin B   against  3 and 6  displaye 3 and 6 . Compounhepsin B . Inactivhepsin B  and comp1 that could explain the irreversible behavior of cathepsin B inhibition, and since RCCs have been reported to be able to generate H2O2 in HTS buffers 2O2. In the redox-free environment H2O2 inhibited cathepsin B as a time-dependent irreversible inhibitor  compound 1 inhibited cathepsin B activity by 60%  the peak shifted to a mass of 28204±1 Da, consistent with the addition of two oxygen atoms and formation of sulfinic acid. To show that this modification is active-site directed cathepsin B was treated with CA-074 (44 µM) prior to addition of compound 1. This resulted in an increase in mass of 28558±1 Da, corresponding to the formation of the covalent adduct between cathepsin B and CA-074 . CA-074 is known to covalently react only with the active site thiol group 1 induces formation of sulfinic acid from the active site thiol group under the experimental conditions used.Additionally, mass spectrometry was used to identify the compound 174±1 Da . After 52O2 is shown to be a major constituent. The inactivation of cysteine cathepsins arises from specific oxidation of the thiol group of the catalytic Cys29 to sulfinic acid.These results demonstrate that 4-aminophenols inactivate cysteine cathepsins B, H and L in a dose- and time-dependent manner. The inhibition is mediated by ROS, of which Ha (∼ 2.5–3.5) a of the thiolate enhances its catalytic function as a nucleophile, however, it also increases its susceptibility to inactivation by ROS, such as H2O22O2 generated by RCCs is detected with horseradish peroxidase mediated oxidation of phenol red ortho-quinones, para-quinones, pyrimidotriazinediones, arylsulfonamides, nitrothiophene-2-carboxylates, tolyl hydrazides, acetamido-dihydroisoxazolopyridine-diyl diacetates and pyrrolo-quinoxalinchlorobenzenamides were revealed Cysteine cathepsins are small monomeric enzymes with molecular weight of approximately 30 kDa and possess a papain-like structure consisting of a left and a right domain. The two domains form a V-shaped active site cleft in the middle of which the catalytic triad is located 1 inhibits cathepsin B in a time-dependent manner. Given that it lacks an electrophilic motif, typically required for irreversible inactivation of the enzyme, and that time-dependent behavior is one of the first indicators of RCCs 1 is seen to be a potent irreversible inhibitor of cathepsin B in redox-free and cystine systems (ortho-quinones (67.8 M−1s−1) 1 was significantly attenuated. These results contrast with reports that redox inactivation by RCCs requires the presence of strong reducing agents like DTT or tris(2-carboxyethyl)phosphine (TCEP) and is not observed in the presence of milder reducing agents, such as glutathione, β-mercaptoethanol, cysteine, or in their absence As part of a previous study we discovered that compound  systems . The sec1 is able to distinguish between individual cathepsins. Cathepsins B, H and L were chosen based on their substrate specificities. Cathepsin H acts as an aminopeptidase, cathepsin L as an endopeptidase and cathepsin B can cleave its substrates both as an endopeptidase and a dipeptidyl carboxypeptidase 1 inhibited all three enzymes within the same concentration range  on range  in non-r1 analogues (compounds 2–8) were also tested for the inhibition of cathepsin B , only 4-aminophenol was able to non-enzymatically induce hydroxyl radical formation A series of compound hepsin B  and S2 tironment  leading 1, 3 and 6) that behaved in a time-dependent manner in the non-reducing environment, only compounds 1 and 3 retained their time-dependent inhibition in a reducing environment  is in line with those for the H2O2 induced inactivation of the related cysteine protease papain (kinact/KI of 61.7 M−1s−1) inact/KI of 10–20 M−1s−1) 2O2 as one of the components of ROS induced by compound 6. H2O2 induces oxidation of the papain active site thiol group to inactive sulfenic acid in the absence of reducing agents, a step which is almost completely reversed by addition of cysteine 2O2, yielding the sulfenic acid of the active site cysteine, which can be reduced back to the active thiolate species with glutathione, DTT, β-mercaptoethanol and cysteine Of the three inhibitors , which is formed form upon further oxidation of sulfenic acid in the second step From these results we can predict the following mechanism for 4-aminophenol induced inactivation of cathepsins . 4-Aminop-benzoquinoneimines with the active site thiol group. This mechanism was proposed to be the main source of hepatotoxicity of acetaminophen. Its metabolic activation leads to the formation of a toxic metabolite, N-acetyl-p-benzoquinoneimine. The latter is preferentially conjugated with glutathione, but once glutathione is depleted the toxic metabolite binds to other nucleophilic groups in the cell, in this way accounting for the toxicity 2O2 formation in cell medium, impairing LLC-PK1 cell viability, in this way exerting its toxicity However, irreversible inactivation of cathepsins by 4-aminophenols can also be explained by a mechanism involving the formation of covalent adducts of To conclude, we report 4-aminophenols as a novel class of RCCs capable of entering the redox cycle, thereby inducing the formation of ROS and producing false positives in enzyme inhibition assays. We propose that compounds with a 4-aminophenol scaffold are eliminated from compound libraries to be screened for inhibition of cysteine proteases. This could be of great importance in HTS campaigns, where the exclusion of false positive hits saves both time and resources otherwise spent on follow up studies.Figure S1Progress curves of cathepsin B activity obtained in the presence of 4-aminophenol analogues in redox-free conditions. 5 µl of DMSO (control) or compounds 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), 8 (H) and H2O2 (I)  and 5 µl of Z-Arg-Arg-AMC (60 µM) were added to wells of a black microplate. Reaction was initiated by adding 90 µl of cathepsin B (400 pM). Formation of fluorescent degradation product was monitored continuously at 380 nm excitation and 460 nm emission wavelengths using the minimal kinetic interval at 37°C. All kinetic measurements were performed in duplicate. For time-dependent inhibitors each progress curve was fitted to the equation st + (vi − vs)(1 − e−kobst)/kobsP  =  v. The obtained obsk were plotted against inhibitor concentration and the slope of linear fit yielded the second-order rate constant inact/KIk.(TIF)Click here for additional data file.Figure S2Progress curves of cathepsin B activity obtained in the presence of 4-aminophenol analogues in the presence of 5 mM cysteine. 5 µl of DMSO (control) or compounds 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), 8 (H) and H2O2 (I)  and 5 µl of Z-Arg-Arg-AMC (60 µM) were added to the wells of a black microplate. The reaction was initiated by adding 90 µl of cathepsin B (400 pM). Formation of fluorescent degradation product was monitored continuously at 380 nm excitation and 460 nm emission wavelengths using the minimal kinetic interval at 37°C. All kinetic measurements were performed in duplicate. For time-dependent inhibitors each progress curve was fitted to the equation st + (vi − vs)(1 − e−kobst)/kobsP  =  v. The obtained obsk were plotted against inhibitor concentration and the slope of linear fit yielded the second-order rate constant inact/KIk.(TIF)Click here for additional data file.Figure S3Binding of CA-074 is not affected by the redox environment. Cathepsin B (1 µg) was incubated with cathepsin B specific irreversible inhibitor CA-074 (200 µM) in a variety of activation buffers containing no reducing agent, cystine, cysteine or DTT (5 mM) and subsequently labeled with DCG-04 (2 µM). Samples were then analyzed by SDS-PAGE and Western blotting. The band intensities indicate cathepsin B activity.(TIF)Click here for additional data file.Figure S42O2 do not significantly impair cathepsin B activity in the presence of 5 mM cysteine, as shown with the DCG-04 probe.Compounds 2–8 and H Cathepsin B (1 µg) was incubated with compounds 2–8 and H2O2 (200 µM) in the activation buffer containing 5 mM cysteine (A) and subsequently labeled with DCG-04 (2 µM). Samples were then analyzed with SDS-PAGE and Western blotting. Band intensities correlate with cathepsin B activity. Residual cathepsin B activity was obtained by dividing individual band intensity by that of the control (B). Data shown here are presented as means ± SD, n = 2.(TIF)Click here for additional data file.Figure S5pI calibration curve. The latter was obtained using the Broad range pI calibration kit (pH 3.0–10.0) according to the protocol of the manufacturer.(TIF)Click here for additional data file.


Heat shock transcription factor 1 (HSF1) plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE) are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications. S. cerevisiae HSF directly activates the expression of genes whose protein products are involved in protein folding and degradation, ion transport, signal transduction, energy generation, carbohydrate metabolism, vesicular transport, cytoskeleton formation and other cellular functions All organisms are exposed to proteotoxic stresses that result in the accumulation of misfolded proteins. In response to these stresses cells have evolved adaptive responses to protect and stabilize cellular proteins until more favorable conditions for cell proliferation are encountered While mammalian cells express four distinct HSF proteins encoded by separate genes, HSF1 is the primary factor responsible for stress responsive gene transcription in vitro phosphorylation experiments and in vivo studies using either lexA or Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain. As such, many of the earlier studies exploring S303 and S307-dependet regulation of HSF1 activity have resulted in conflicting results. For example, previous phosphorylation experiments suggested that S307 was phosphorylated by ERK which, in turn, acted as an essential priming step for GSK3-dependent phosphorylation of S303 in vitro studies suggested that S303 could also be phosphorylated by a variety of mitogen activated protein kinases (MAPK) including the stress responsive MAPK p38 in vivo data suggested S303 phosphorylation could occur independently of S307 phosphorylation The activity of HSF1 is also thought to be negatively regulated through a number of post-translational modifications including phosphorylation, sumoylation and acetylation While the specific mechanism by which S303 and S307 phosphorylation repress HSF1 activity remains unclear, evidence has suggested that S303 and S307 phosphorylation represses the transactivation potential of HSF1 S. cerevisiae is unable to complement for the loss of the essential yeast HSF protein While HSF1 and the cognate HSEs are quite well conserved from yeast to humans, our previous results demonstrated that human HSF1 expressed in Here we report the use of the yeast assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. Our results suggest that S303 phosphorylation blocks human HSF1 homotrimerization thereby preventing human HSF1 activation and complementation of the loss of yeast HSF. Furthermore, we demonstrate that S303 phosphorylation also blocks HSF1 homotrimerization in mammalian cells. We show that phosphorylation of HSF1 S303 in yeast occurs via the action of the MAPK Slt2 and not via the action of GSK3 and we extend these findings to show that S303 phosphorylation also occurs independent of GSK3 in mammalian cells.When human HSF1 is expressed in yeast it is unable to homotrimerize, promote gene expression and complement for the loss of the essential yeast HSF protein To ascertain whether HSF1 is being phosphorylated in yeast, we employed a commercially available antibody specific for phospho-S303 (pS303). Because this antibody has not previously been characterized in the literature, we tested its specificity in human HeLa cells where S303 is known to be constitutively phosphorylated Consistent with a contribution to HSF1 repression  S303 is Human HSF1 S303 phosphorylation is known to promote sumoylation of lysine 298, which also contributes to the repression of HSF1 activity −/− mouse embryonic fibroblasts (MEF) Previous reports have suggested that phosphorylation of S303 represses the ability of HSF1 to transactivate gene expression In addition to post-translational modifications, HSF1 activity is also thought to be repressed through intramolecular interactions between carboxyl- and amino-terminal coiled-coil domains and mutations in these domains render HSF1 constitutively trimerized, nuclear localized and bound to DNA in mammalian cells in vitro phosphorylation experiments have suggested that HSF1 is phosphorylated at S303 by glycogen synthase kinase 3 (GSK3) in vivo. To test if GSK3 contributes to HSF1 repression, we assayed human HSF1-dependent yeast growth in a strain also lacking the yeast GSK3 homolog Rim11. Supporting the notion that yeast GSK3 can repress human HSF1 activity in yeast we observed human HSF1-dependent yeast growth as well as HSF1 multimerization in the rim11Δ strain  in yeast phosphorylate human HSF1 at S303 to promote HSF1 repression, we assayed S303 phosphorylation in several previously generated protein kinase deletion strains obtained from the yeast gene deletion collection e strain . Homotriin yeast . In mammΔ strain  also supin vitro phosphorylation experiments in vivo using lexA/Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain −/− MEFs which lack endogenous HSF1. When we expressed S303A, S307A or S303/307A HSF1 mutants in hsf1−/− MEFs we observed a modest elevation of Hsp70 expression under normal growth conditions  supplemented with protease inhibitors (Roche) and Halt phosphate inhibitor cocktail (Thermo Scientific Pierce). Proteins extracts were generated from mammalian cell culture using cell lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA assay and 80–100 µg of total protein was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. HSF1 oligomerization was assessed using the amine-specific cross-linker ethylene glycol bis-succinimidyl succinate (EGS) (Pierce). Crosslinking analysis were carried out as described previously


Euphausia crystallorophias and Antarctic krill E. superba), fish  and seabirds . This study provides the first evidence of the incorporation of HBI lipids in Antarctic pelagic consumers. Specifically, a di-unsaturated HBI (diene) of sea ice origin was more abundant in ice-associated species than in pelagic species, whereas a tri-unsaturated HBI (triene) of phytoplanktonic origin was more abundant in pelagic species than in ice-associated species. Moreover, the relative abundances of diene and triene in seabird tissues and eggs were higher during a year of good sea ice conditions than in a year of poor ice conditions. In turn, the higher contribution of ice algal derived organic matter to the diet of seabirds was related to earlier breeding and higher breeding success. HBI biomarkers are a promising tool for estimating the contribution of organic matter derived from ice algae in pelagic consumers from Antarctica.The structure, functioning and dynamics of polar marine ecosystems are strongly influenced by the extent of sea ice. Ice algae and pelagic phytoplankton represent the primary sources of nutrition for higher trophic-level organisms in seasonally ice-covered areas, but their relative contributions to polar marine consumers remain largely unexplored. Here, we investigated the potential of diatom-specific lipid markers and highly branched isoprenoids (HBIs) for estimating the importance of these two carbon pools in an Antarctic pelagic ecosystem. Using GC-MS analysis, we studied HBI biomarkers in key marine species over three years in Adélie Land, Antarctica: euphausiids (ice krill  Shrinking sea ice threatens the structure, functioning and dynamics of polar marine ecosystems 25 HBIs are synthesised by a few diatom species 13C enriched relative to the more unsaturated HBIs Haslea and/or Navicula species of diatoms, which live in the brine channels at the base of Antarctic sea ice and bloom in spring 13C values of triene and other poly-unsaturated isomers are consistent with a phytoplanktonic origin Rhizosolenia spp., are likely responsible for the synthesis of triene and other poly-unsaturated HBI isomers versus phytoplankton biomarkers in polar marine ecosystems. Three recent studies have detected HBI isomers in pelagic zooplankton and benthic macrofauna in the Arctic The aim of the present study was to evaluate the potential of some novel lipid markers, highly branched isoprenoids (HBIs), for estimating the contribution of organic matter derived from ice algae in pelagic consumers from Antarctica. CHere, we investigated the potential of this approach for studying the Antarctic pelagic ecosystem. Our first aim was to determine whether HBIs are transferred to key pelagic species in Antarctica. We collected specimens of two krill species, two planktivorous fish species and three seabird species and analysed the presence and concentrations of HBIs. Our second aim was to identify whether the sea-ice-specific diene was more abundant in species preferentially feeding in ice-covered waters (cryopelagic species) than in pelagic species by comparing corresponding tissues among closely related taxa. Finally, we tested (i) whether Antarctic seabirds incorporate more diene during a year of extended pack ice than during a year of reduced ice and (ii) whether a high contribution of ice algal-derived organic matter predict key ecological parameters; i.e., earlier breeding and higher reproductive success.Ethical approval for all procedures was granted by the ethics committee of the Ministère de l'Environnement and the French Polar Research Institute (Institut Paul Emile Victor – IPEV), which approved all our fieldwork. The experiments complied with the Code of Ethics of Animal Experimentation in the Antarctic.Pagothenia borchgrevinki were studied in the Pointe Géologie Archipelago  and the Antarctic silverfish Pleuragramma antarcticum were collected from the Research Icebreaker Astrolabe using an Isaacs-Kidd Midwater Trawl (IKMT). The Adélie Land area is ice covered seasonally, with land-fast ice typically forming in March/April and breaking up in November/December. The cruise was conducted in January 2011 in open waters , 870 pairs of snow petrels (Pagodroma nivea) and 450 pairs of cape petrels (Daption capense) th of December to the 21st of January) did not differ between 2009/2010 and 2010/2011 . All samples were immediately frozen at −80°C until analysis at the LOCEAN/IPSL  laboratory.Pointe Géologie Archipelago is a breeding area for approximately 36,000 pairs of Adélie penguins  to yield an apolar lipid fraction containing the HBIs. This hydrocarbon fraction was analysed using an Agilent 7890A gas chromatograph (GC) fitted with a 30 m fused silica Agilent J&C GC column  coupled to a Agilent 5975C Series mass selective detector (MSD). A ramped oven temperature profile  followed by an isothermal interval (300°C for 10 min) was used. Data were collected and analysed with Agilent Chemstation software. Individual compounds were identified and quantified using both scan (m/z 50–500 mass range) and selective ion monitoring (SIM) techniques with a constant ionisation potential (70 eV). Mass spectral data were used to identify the occurrence of HBIs, whereas SIM chromatograms were used to quantify the abundances of diene (m/z 348.3), triene (m/z 346.3), tetraene (m/z 344.3) and pentaene (m/z 342.3) by peak area integration. Individual HBI isomers were identified by comparing their GC retention indices and mass spectra with those of previously authenticated standards. HBI relative abundances were calculated based on their individual GC-MS responses and those of the internal standard . As we did not determine their individual response factors, HBI abundances were expressed in ng.g−1 relative to the internal standard. Procedural blanks were analysed for each 20 samples to ensure the absence of contamination.HBI analyses were conducted according to a previously described procedure In the laboratory, chick stomach contents were placed in a large flat-bottomed tray. Sixteen stomachs contained prey items that were not completely digested. The most abundant prey items were identified to species in three to six sub-samples of each food sample. Prey were identified through examination of exoskeletons (for crustaceans) and of otoliths and bones (for fish). Preys items were counted in each sample. We estimated the proportions of fresh mass represented by each species.ftp://ftp-projects.zmaw.de/seaice/AMSR-E_ASI_IceConc/no_landmask/hdf/s6250/Daily remote-sensing sea ice concentration maps, retrieved from the Advanced Microwave Scanning Radiometer–Earth Observing System (AMSR-E), were obtained from the University of Hamburg, Institute of Oceanography from the web site Phenological and reproductive success data were obtained for seabirds via long term monitoring programs conducted by the Centre d'Etudes Biologiques de Chizé All statistical analyses were performed using R 2.14.2 −1 and the abundance of triene varied from 0.53 to 473 ng.g−1 both relative to the internal standard. It is important to notice that HBI analyses were conducted either on whole organisms or biological tissues on dissected organisms. The relative abundances of diene and triene were the highest in krill and in stomach contents/oils, livers, eggs of seabirds, whereas diene and triene were in very low abundance in muscles of Adélie penguins and fish, kidneys, adipose tissues, and blood of Adélie penguins. Moreover, concentrations of diene varied greatly between tissue type in Adélie penguin chicks . The highest concentrations of HBIs in Adélie penguin were detected in the stomach content, followed by eggs and liver. The lowest concentrations were found in the quadriceps, pectoralis and gastrocnemius muscles, kidneys, subcutaneous and retroperitoneal white adipose tissues and blood.Diene and triene were detected in all Antarctic pelagic organisms, including krill, fish and seabirds . The abuD/T ratios did not differ between Antarctic and ice krill but differed significantly between sampling sites but eastern sites (i.e. AS607 and AS608) having significantly higher D/T ratios than western sites , sea ice concentrations were significantly higher in 2009/2010 (mean ± SE during these 3 months: 55.8%±2.1%) than in 2010/2011  to the 21st of January (30-day old). During this sampling period, sea ice concentrations were also significantly higher in 2009/2010 (37.2%±1.2%) than in 2010/2011 .During the Adélie penguin breeding season  and in stomach contents of Adélie penguin chicks were significantly higher during the 2009/2010 breeding season than during the 2010/2011 breeding season  and more consumption of Antarctic krill  in 2009/2010 than in 2010/2011 . The meaThe long term monitoring programme conducted on Petrels Island, where Adélie penguin dead chicks were collected, revealed that the total number of Adélie penguin pairs that bred did not differ significantly between years (15999 breeding pairs in 2009/2010 and 14694 in 2010/2011). However, both the number of chicks in crèche  and the number of fledglings  were higher in 2009/2010 than in 2010/2011. Egg-laying date (peak: 17/11/2009 and 22–23/11/2010) and hatching date (peak: 21/12/2009 and 23–24/12/2010) were approximately 2.5 days earlier in 2009/2010 than in 2010/2011.We investigated HBI concentrations in abandoned snow petrel eggs between 2007 and 2010. Female snow petrels forage at sea from mid-November to mid-December while forming their single eggs. Analysis of satellite data revealed that sea ice concentrations were significantly higher in 2007 than in 2010 during this period (t = 5.647, df = 28, p<0.001, HBI markers have recently been identified in Arctic benthic macrofauna and pelagic zooplankton In Adélie penguin chicks, we observed large differences in the concentrations of HBIs between tissue types, suggesting a time-integrated assimilation of HBI lipids within organisms as for fatty acids Krill use both ice and pelagic food sources throughout the year D/T ratios were higher in muscles of bald notothen collected under the ice than in muscles of Antarctic silverfish collected in open waters. The bald notothen feeds primarily on cryopelagic copepods, pteropods, gammarids and hyperiids in platelet ice and water beneath the ice At the top of the trophic web, D/T ratios within chick stomach oil were higher in snow petrels, which are piscivorous seabirds confined to the pack ice zone To control for among-tissue differences, we performed comparisons on the same tissue types of Adélie penguins during the two consecutive breeding seasons. D/T ratios in all tissues and stomach contents of Adélie penguin chicks were lower in 2010/2011 than in 2009/2010, coinciding with 10% lower daily sea ice concentrations during the 2010/2011 breeding season than in the 2009/2010 breeding season. Although little is known about HBI production and D/T ratio changes through time in Antarctica, these results suggest a reduced contribution of ice algal-derived organic matter in the diet of chick-rearing Adélie penguins in 2010/2011. Furthermore, prey items in chick stomachs confirmed a change in diet between years. Although ice krill was the predominant prey (as reported elsewhere; e.g., Annual variation in the phenology and ecology of Antarctic top predators with variation in the extent of sea ice has been widely discussed


We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24–48 h. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components  confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), improves insulin sensitivity in acute studies  Rapamycin is the only drug that has been unequivocally established to robustly and reproducibly extend maximum lifespan in mice . HoweverThe canonical target of rapamycin is mechanistic  target of rapamycin complex 1 (mTORC1), a kinase involved in nutrient sensing and the regulation of growth and metabolism . The mTOFigure 1; in vivo, consistent with our biochemical demonstration that mTORC2 can be disrupted in intact tissues in mice.We have shown that rapamycin-induced insulin resistance in mice is at least partially due to disruption of mTORC2, a second mTOR-containing complex that phosphorylates several substrates, including AKT at serine 473 as part of the insulin signaling cascade  improvement in insulin responsiveness correlates to the acute loss of signaling through S6K1, consistent with disruption of the negative feedback loop driving inhibitory phosphorylation of IRS1 (Antibodies to phospho-AKT S473 (9271), phospho-AKT T308 (9275), AKT (9272), phospho-S6 ribosomal protein (2215), S6 ribosomal protein (2217), mTOR (2972), Raptor (2280), Rictor (2140), phospho-PKC theta T538 (9377), and VDAC (4866) were from Cell Signaling Technology. The phospho-IRS1 S307 (07-247) antibody was from Upstate and IRS1 (05-1085) and GAPDH (JC1641540) antibodies were from Millipore. OXPHOS cocktail (MS604/D1848) was from MitoSciences. Protease and phosphatase inhibitor cocktail tablets were from Roche . Rapamycin was purchased from Calbiochem (553210). Torin1 was a gift from the lab of Nathanael Gray. Triglyceride reagent was from Stanbio Laboratory. DMEM, fetal bovine serum (FBS), horse serum, insulin, protein A agarose, and Trizol were obtained from Invitrogen. Other chemicals were purchased from Sigma unless noted.°C for 10 min. The complexed palmitate was then diluted to a final concentration of 0.5 mM in DMEM containing 2% BSA, cooled at RT, filtered and administrated to C2C12 myotubes for 16 h.C2C12 mouse myoblasts were maintained in DMEM supplemented with 10% FBS and antibiotics (Pen/Strep). When cells reached confluence, the medium was switched to DMEM with 2% horse serum. Cells were refreshed with differentiation medium every other day. By day 4, C2C12s were fused into myotubes. C2C12 myotubes were then treated with 500 nM rapamycin for the indicated times. Torin1 treatment was at 250 nM for 4 h. Palmitate-containing media were prepared by conjugating palmitate with FFA-free bovine serum albumin as described by 2 incubator, as recommended by Seahorse Biosciences. A Seahorse XF24 Analyzer (Seahorse Biosciences) was then used to measure the oxygen consumption rate. Each cycle included 4 min of mixing, a 2-min wait, and then measurement over 2 min. One micromolar oligomycin was used to inhibit ATP synthase. Next, 300 nM FCCP was added to stimulate uncoupled respiration. Immediately after measurement, the protein concentration was measured by bicinchoninic acid (BCA) assay (Pierce Biotechnology).C2C12 myoblasts were seeded at 40,000 cells per well in 24-well XF plates. Myoblasts were treated with rapamycin for the indicated times, or alternatively, myotubes were generated by replacing the medium with differentiation medium when cells reached confluence. After an 4 days of differentiation, myotubes were treated with rapamycin for the indicated times. The cells were refreshed with non-buffered pH 7.4 medium (25 mM glucose) and incubated for 1 h in a non-CO°C. Protein concentration was determined by BCA assay (Pierce Biotechnology). Twenty microgram proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8–16% gradient or 7.5% resolving gels. Quantification was performed by densitometry using ImageJ software, and loading was verified by blotting for GAPDH.Cells were rinsed with PBS and lysed in cold RIPA buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets. Cell lysates were incubated on ice for 10 min, sonicated on ice for 30 s, and centrifuged at 12,800 rpm for 15 min at 4°C for 15 min and then centrifuged at 16,000 rpm for 15 min at 4°C to remove insoluble material. Protein A agarose beads were added to the supernatant and incubated with rotation for 1 h. Next, the beads were centrifuged out of the lysates and mTOR or Rictor antibodies were added to the cleared lysates, which were rotated overnight at 4°C. Protein A agarose beads were added to the supernatant and incubated at 4°C for an additional hour. Immunoprecipitated complexes with protein A agarose beads were washed in 0.3% CHAPS lysis buffer three times, boiled in SDS-sample buffer, separated by SDS-PAGE (7.5% gels), and analyzed by immunoblotting. This method is adapted from that described by C2C12 myotubes were lysed in cold 0.3% CHAPS lysis buffer . Cells lysates were incubated at 4Total RNA was extracted from the sample using TRIzol reagent. The concentration and purity of RNA were determined by absorbance at 260/280 nm. One microgram of total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions. The cDNA was subjected to real time PCR using SYBR Q-PCR master mix (Applied Biosystems). Primer sequences used to produce gene-specific amplicons are as follows: PGC-1α: forward: ACTATGAATCAAGCCACTACAGAC; reverse: TTCATCCCTCTTGAGCCTTTCG, GAPDH: forward: GGTGAAGGTCGGAGTCAACGGA; reverse: GAGGGATCTCG- CTCCTGGAAGA, TFAM: forward: AAGACCTCGTTCAGCATA- TAACATT; reverse: TTTTCCAAGCCTCATTTACAAGC, A typical reaction contained 250 nM of forward and reverse primer, 1 μl cDNA and the final reaction volume was 20 μl. The reaction was initiated by preheating at 50°C for 2 min, followed by 95°C for 10 min. Subsequently, 40 amplification cycles were carried out with 15 s denaturation at 95°C and 30 s annealing and extension at 60°C. Gene expression was normalized to GAPDH.°C. 170 μl of 5 M NaCl was added and samples were mixed for 1 min and then centrifuged at max speed for 15 min at 4°C. Supernatants were collected and 1 ml ethanol was added, after which the tubes were inverted several times. Samples were centrifuged at max speed for 15 min at 4°C, after which ethanol supernatant was aspirated and DNA pellet was washed in 500 μl of 70% ethanol. Samples were centrifuged for 5 min, max speed at 4°C, ethanol supernatant was removed, the DNA pellet was air dried and resuspended in 50 μl of TE. Primer sequences used to produce mitochondrial and nuclear specific DNA products for quantification of mtDNA/nuclear DNA ratios are as follows: MT-CO1: forward: TGCTAGCCGCAGGCATTAC; reverse: GGGTGCCCAAAGAATCAGAAC, Ndufv1: forward: CTTCCCCACTGGCCTCAAG; reverse: CCAAAACCCAGTGATCCAGC.Briefly, cell pellets were digested in with 15 μl proteinase K (10 mg/ml) in a 500 μl total volume of proteinase K buffer  overnight at 55293T cells were from ATCC and were seeded onto 10 cm diameter dishes with DMEM (supplemented with 10% FBS and penicillin–streptomycin) and incubated overnight. When the cells became 70% confluent, they were refreshed the cells with antibiotic-free DMEM. Next, the Rictor shRNA, Raptor shRNA, and control GFP shRNA plasmids , togetheC2C12 myotubes were lysed in PBS containing 1% Nonidet P-40. Triglycerides were determined by triglyceride reagent according to manufacture’s instructions (Stanbio Laboratory). Intracellular triglycerides were normalized to total protein content.t-test in Microsoft Excel  or a one-sample t-test to test whether ratios of phosphorylated Akt differed significantly from the control value (set to one) in Figures 6. * denotes p < 0.05 compared to control unless otherwise indicated.Densitometry was performed using ImageJ software  and band intensities were normalized to GAPDH, unless otherwise indicated. Significance was tested using the two-tailed unpaired Student’s We initially chose to explore the effects of rapamycin in differentiated C2C12 myotubes cultured in the presence of palmitate because it is known that insulin sensitivity in this system is decreased by the accumulation of lipid intermediates, such as diacylglycerols and ceramides, and that enhancing mitochondrial fatty acid oxidation is sufficient to improve insulin sensitivity . TherefoFigure 2). Over four independent experiments, the average effect of short-term rapamycin was an approximately 50% increase in the pAkt:Akt ratio following insulin stimulation, p = 0.02. Interestingly, when the rapamycin pre-treatment was extended to 48 h, the myotubes became almost completely unresponsive to insulin stimulation, consistent either with further antagonism of lipid-induced insulin resistance via inhibition of mitochondria, or with disruption of mTORC2. Over four independent experiments, the average effect was a 67% decrease in the pAkt:Akt ratio, p < 0.001. AICAR, an AMPK activator that promotes respiration and mitochondrial biogenesis , and uncoupled conditions . To test whether rapamycin might have changed the substrate preference of the mitochondria, we also added palmitate to the media. However, the addition of a fatty acid substrate did not restore respiration rates in the rapamycin-treated cells, and had only a minor effect on oxygen consumption, which was reversed upon addition of the carnitine palmitoyl transferase 1 (CPT-1) inhibitor etomoxir. Intriguingly, a significant reduction in oxygen consumption occurred after 1 h of pre-treatment, suggesting that it was largely due to changes in flux through the existing mitochondria, rather than to a reduction in the rate of mitochondrial biogenesis.To test whether rapamycin was sufficient to suppress mitochondrial function, we measured respiration rates in treated or untreated myotubes using a Seahorse XF24 Flux Analyzer. As shown in Figure 4A; Figure 4B), we did not detect any significant effect of rapamycin on mitochondrial DNA content or the levels of various mitochondrial proteins over the time course of this experiment . Lipid-induced insulin resistance is typically associated with increased phosphorylation of atypical PKC isoforms and inhibitory (serine) phosphorylation of IRS1. Serine phosphorylation of IRS1 was not enhanced. In fact, IRS1 phosphorylation was diminished in the presence of rapamycin at all time points studied, likely reflecting the inhibition of S6K1. Phosphorylated (active) PKCθ , was clearly higher in the presence of palmitate, and appeared to be incrementally increased by rapamycin. However, there was no induction of PKCθ phosphorylation by rapamycin in the absence of palmitate, yet the myotubes still became insulin resistant . Together, these observations strongly suggested that mitochondrial dysfunction, leading to lipid-induced insulin resistance is not the major mechanism accounting for reduced insulin sensitivity in rapamycin-treated myotubes.Next, we examined factors related to mitochondrial biogenesis and lipid-induced insulin resistance in rapamycin-treated myotubes. Although we confirmed the previously reported suppression of mitochondrial transcription factors . These results could account for the loss of AKT phosphorylation at serine 473, which appears to be required for subsequent phosphorylation by PDK1 at threonine 308 in many cases  or the mTOR catalytic subunit . On the other hand, Rictor knockdown significantly diminished AKT phosphorylation at serine 473 as well as threonine 308, confirming that this site is sensitive to mTORC2 inhibition in C2C12 myotubes . Torin1, a direct inhibitor of both mTORC1 and mTORC2 , although it should be noted that inhibition of upstream signaling through PI3K may have contributed to the Torin1 result . However, this mechanism may play a role in the longer-term effects of rapamycin on insulin sensitivity in skeletal muscle in vivo.Interestingly, a third potential mechanism by which rapamycin could cause insulin resistance in skeletal muscle was recently described by in vivo. Rapamycin binds directly to FKBP12 and FKBP12.6, and inhibits mTORC1 via a drug-induced interaction (in vivo consequences of rapamycin treatment may include effects that go beyond the canonical inhibition of mTORC1 and the disruption of mTORC2.It is also intriguing to speculate that rapamycin may have effects that are independent from the mTOR complexes, in cells and eraction . FKBP12 eraction  and neureraction  phenotyperaction . Therefoin vivo.Rapamycin is the only drug that has been proven to extend the maximum lifespan of a mammalian species in a rigorous, multi-center trial using lean, healthy animals that are not predisposed to any specific disease . In contThe 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.


No matter what you're doing at any given moment, from walking to talking or evensleeping, your brain is doing its own thing. Networks of neurons constantly andoften spontaneously generate rhythmic electrical activity in the cortex, thebrain's outermost layer and the seat of judgment, decision making, and otherhigher order functions. These cortical networks contain pyramidal cells that canexcite inhibitory interneurons, which can in turn decrease the activity of pyramidalcells. This interplay of excitation and inhibition generates specific activitypatterns that are critical to various cortical functions, like working memory andattention. However, it is not clear what cellular mechanisms maintain the properbalance between these two opposing inputs.PLoS Biology, Yousheng Shu and colleaguesreport that small changes in the electrical properties of pyramidal cells helpmaintain the excitation–inhibition balance that keeps these cortical networkshumming along. In addition to exciting other neurons via “all or none”events  called action potentials, pyramidal cells mayalso have another way of communicating within a network. The researchers hadpreviously found that pyramidal cells can use a “graded” method  of exciting their targets via small changes in their membrane potential.Because pyramidal cells activate inhibitory interneurons and thus generate recurrentinhibition, the researchers asked whether this analog control of membrane potentialcould fine-tune the balance between excitation and inhibition in the cortex.Now, in this issue of The researchers began investigating recurrent network activity by recording activitybetween pairs of nearby pyramidal cells that presumably had an inhibitoryinterneuron between them. They first established that electrically stimulating onepyramidal cell in this microcircuit resulted in a late-onset, “slow”recurrent inhibition in the second pyramidal cell. They next made a positive(depolarizing) shift of the membrane potential by injecting current into the firstpyramidal cell, and found that this increased the slow recurrent inhibition in thesecond cell. This modulation was sensitive to membrane potential shifts as small as5 to 10 mV, considerably less than the shifts required to generate an all-or-noneaction potential.What about the other connections in this microcircuit? Knowing that low-thresholdspiking (LTS) interneurons can mediate slow recurrent inhibition, the authors nextasked whether modulation of pyramidal cells can directly influence these inhibitorycells. Indeed, they found that small membrane potential shifts in pyramidal cellscan modulate LTS interneuron activity. Importantly, they also observed these analogeffects for fast spiking interneurons, which mediate “fast” recurrentinhibition. Taken together, these findings demonstrate that the membrane potentialof pyramidal cells modulates recurrent inhibition, which helps balance theexcitation and inhibition that lend stability to cortical network rhythms.Finally, the researchers examined the possible mechanisms of this membrane potentialeffect, and found a role for a type of potassium current called the D-current thathelps control the duration of axonal action potentials. Blocking the D-current withdrugs increased the inhibitory effect between pairs of pyramidal cells as well asthe excitatory effect of pyramidal cells on LTS interneurons. Based on thesefindings, the researchers proposed the following model: depolarization in the firstpyramidal cell inactivates D-current and so prolongs axonal action potentials,thereby enhancing synaptic transmission to the interneuron that then causes moreinhibition to the second pyramidal cell.By showing that membrane potential helps balance excitation and inhibition inmicrocircuits, this work suggests a key role of analog communication in the rhythmicactivity of cortical networks. Notably, recent work has implicated disruptions inthe balance of excitation–inhibition in neurological disorders such asepilepsy and schizophrenia. Whether analog modulation may prove relevant to suchdiseases, however, is unknown. Many questions must be investigated before suchpossibilities can be addressed, including whether analog modulation applies to allcortical circuits, and whether it occurs during behaviorally relevant processes.Zhu J, Jiang M, Yang M, Hou H, Shu Y (2011) Membrane Potential-DependentModulation of Recurrent Inhibition in Rat Neocortex.doi:10.1371/journal.pbio.1001032


The protein kinase activity of PI3K phosphorylates specific serine residues in growth factor receptors to promote cell survival; these events are constitutively activated in some leukemias. The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer. The ability of cells to survive in the absence of proliferation (cell division), differentiation (cell maturation) or activation allows tissues to maintain cell populations that are poised for rapid responses to damage, infections, or other physiological demands. While this “survival-only” response is fundamental to all physiological processes, the underlying mechanisms are not understood. Many growth factors are potent regulators of cell survival through their ability to bind specific cell surface receptors, which in turn activate specialized enzymes called kinases. Phosphoinositide 3-kinase (PI3K) is a dual specificity kinase that is known to be involved in cell survival and malignant transformation, and it is able to phosphorylate both lipid and protein substrates. While the PI3K lipid kinase activity has been extensively studied, the functional significance of its protein kinase activity remains unclear. Here we show that PI3K protein kinase activity can directly phosphorylate growth factor receptors on human hematopoietic (blood) cells to promote a “survival-only” response. We further show that the protein kinase activity of PI3K can be hijacked to result in uncontrolled growth factor receptor phosphorylation and the deregulated survival of leukemic cells. Our studies provide the first evidence that the protein kinase activity of PI3K can control cell survival and that this activity may be deregulated in cancer. A key mechanism by which growth factors and cytokines promote cell survival is via the phosphoinositide 3-kinase (PI3K) pathway and constitutive PI3K signaling is known to promote autonomous cell survival and transformation We and others have shown that the phosphorylation of specific serine residues in the cytoplasmic tails of growth factor and cytokine receptors is critical for initiating intracellular signaling pathways that selectively control cell survival We therefore sought to identify the kinases that promote cellular transformation through their ability to constitutively phosphorylate serine residues in cytokine receptors. Using primary human AML patient samples, we have isolated a kinase that phosphorylates Ser585 in the cytoplasmic tail of the GM-CSF/IL-3 βc receptor. We have identified this Ser585 kinase as the p110α catalytic subunit of PI3K and show that physiological picomolar concentrations of cytokine activate the protein kinase activity of PI3K leading to Ser585 phosphorylation and cell survival. Inhibition of p110α using pharmacological and RNA interference approaches reduced Ser585 phosphorylation in multiple cell types including primary human AML blasts whereas expression of a mutant form of p110α that was lipid kinase-defective but protein kinase-active restored Ser585 phosphorylation. Our findings identify a new role for the protein kinase activity of PI3K in promoting cytokine-mediated cell survival and provide a novel functional link between the deregulated PI3K protein kinase activity and phosphotyrosine-independent survival programs in leukemia.GM-CSF and IL-3 receptor signaling regulate both proliferation and survival of normal myeloid cells and play an important role in myeloid leukemia In order to identify the kinases responsible for phosphorylating Ser585 and promoting cell survival we performed chromatographic fractionation of an AML patient sample exhibiting constitutive Ser585 phosphorylation . Eluted ++ and DTT were present  While the ability of PI3K to promote cell survival has almost exclusively been attributed to its lipid kinase activity, the potential biological roles of PI3K protein kinase activity remain unknown. Our previous studies have shown that very low cytokine concentrations in the picomolar range can promote the phosphorylation of Ser585 within the GM-CSF and IL-3 βc receptor to promote cell survival in the absence of both phosphotyrosine pathways and proliferation We then examined whether PI3K lipid kinase activity was essential for regulating cell survival by examining the role of the key downstream lipid signaling target of PI3K, Akt. Our results show that there was no significant defect in the ability of 1 pM murine GM-CSF to promote the survival of primary mouse bone marrow (BM) progenitor cells derived from either Akt1−/− mice , left orWe next examined the ability of low picomolar cytokine concentrations to promote cell survival under conditions where the protein kinase activity of PI3K was blocked (using the YM024 or PIK-75 p110α-selective PI3K inhibitors) while downstream PI3K lipid signaling was enforced by expression of myr-Akt-1. Blockade of PI3K protein kinase activity induced by 1 pM GM-CSF using either YM024 or PIK-75 resulted in cell death despite constitutive signaling by myr-Akt-1 suggesting that the protein kinase activity of PI3K was required for cell survival and could not be rescued by enforced Akt1 signaling  and S3B.We then examined whether constitutive activation of the protein kinase activity of PI3K was able to promote Ser585 phosphorylation of the endogenous βc subunit of the GM-CSF receptor and cytokine-independent cell survival. For these experiments we utilized a doxycycline-inducible system for the expression of a cytokine-independent membrane-localized form of p110α with both lipid and protein kinase activity (p110α-CAAX) or only protein kinase activity (p110α-4KA-CAAX). Induction of p110α-CAAX using doxycycline in the absence of cytokine resulted in increased Ser585 phosphorylation as well as downstream Akt phosphorylation, both of which were blocked by the YM024 PI3K inhibitor . ImportaWe next examined the impact of inhibiting PI3K on Ser585 phosphorylation and cell survival. As shown in p = 0.001, Mann-Whitney U).We then screened a panel of siRNAs for their ability to knockdown p110α in HEK 293T cells and examined the impact on Ser585 phosphorylation. As shown in We then tested the ability of YM024 and PIK-75 to induce apoptosis in AML blasts derived from patient samples that were sensitive to down-regulation of Ser585 phosphorylation following PI3K inhibition. Our results show that YM024 and PIK-75 were able to induce cell death in primary AML blasts whereas inhibition of p110β (TGX-221), p110δ (IC87114), and p110γ (AS252424) were less effective  and S5B.While many cytokines and growth factors are able to regulate PI3K lipid signaling, little is known of their ability to regulate PI3K protein kinase signaling or whether the protein kinase activity of PI3K is also important in promoting cellular responses in certain contexts. Previously, we and others have identified key serine residues in the cytoplasmic tails of cytokine and growth factor receptors that selectively control cell survival While the lipid kinase activity of PI3K is clearly pivotal in regulating a wide array of cellular responses including cell survival The activation of canonical Type 1A PI3K lipid signaling requires the recruitment of p85 SH2 domains to pYXXM (where pY is phosphotyrosine) phosphotyrosine docking sites, either in the cytoplasmic tails of cell surface receptors or their associated signaling proteins Others have also suggested that PI3K can provide multiple independent signaling outputs with p110γ regulating Akt signaling via its lipid kinase activity and regulating ERK signaling via its protein kinase activity It is important to note that siRNA-mediated knockdown of p110α or pharmacological inhibition inhibits both the protein and lipid kinase activity of PI3K. Thus, the induction of apoptosis following PI3K inhibition may not only result from inhibition of PI3K protein kinase targets (such as Ser585), but also lipid kinase targets (such as Akt) , model. Others have shown that targeting p110δ with IC87114 prevents the proliferation of AML blasts, but the effect on the cell survival has not been determined 579LGPPHSRSLPDILG591 and 579LGPPHSRpSLPDILG591 (where pS is phospho-Ser585 which was used as a non-phosphorylatable control). Recombinant purified p110α was from Meredith Layton (Monash University). Murine GM-CSF and IL-3 were from Prospect. BM from Akt1−/− knockout mice were from Rick Pearson . Akt1-PH domain plasmid obtained from Christina Mitchell (Monash University). A CAAX box was engineered into the C-terminus of p110α by PCR amplification of a 3′ fragment from pcDNA3.1-myc-p110α using GCGGCCATCGATTTGTTTACAC and TTTCGCGCGGCCGCTCAAGAGAGCACACACTTACAGTTCAAAGCATGCTGCTTAA and cloned into the Cla1/Not1 sites of pcDNA3.1-myc-p110α and pcDNA3.1-myc-p110α-4KA , which expresses a mutant form of p110α in which lysines 941–944 within the lipid binding pocket are mutated to alanine, which results in defective lipid kinase activity while protein kinase activity is unaffected. The full length myc-p110α-CAAX cDNAs were then PCR amplified using GAGGAGGACCTGCTGCCTCCAAGACCATCATCAGGTGAACTG and GAACTGTAAGTGTGTGCTCTCTTGAAGCGCTCCGAAA followed by PCR using AAACGGACCGGTGCCACCATGGAGCAGAAGCTGATCTCCGAGGAGGACCTGCTGCCTC and TTTCGGAGCGCTTCAAGAGAGCACACACTTACAGTTC and the products cloned into pTripz using Age1 and Afe1 to give pTripz-myc-p110α-CAAX and pTripz-myc-p110α-4KA-CAAX.Bisindolylmaleimide I, rapamycin, LY294002, GSK-3 inhibitor IX, JAK inhibitor 1 (JAKI), U0126, quercetin, PI3Kγ-1, genistein, PI-103, TGX-221, and AS252424 were from Calbiochem; SB203580 from Promega; H89, PP1, staurosporine, and kemptide from Biomol; imatinib and dasatinib were from Selleck Chemicals; Akt inhibitor 1 was from MBL; 5,6-dichlorobenzimidazole riboside (DRB), AG1296, and Wortmannin were from Sigma; CEP-701 was from Tocris Biosciences; IC87114 and YM024 were generously provided by Shaun Jackson (ACBD). PIK-75 and A66 were synthesized as previously described −) cells were isolated by negative selection using a Lineage Cell Depletion Kit (Miltenyi Biotec) HEK 293T cells were transfected with using lipofectamine (Invitrogen) in 0.5% fetal calf serum  and DMEM for 4 h. TF-1 factor-dependent cell line was cultured in 10% FCS/RPMI with 2 ng/ml human GM-CSF and transfected by electroporation . FDM cell lines were generated by HoxB8 transformation as described in Apheresis product, BM, or peripheral blood samples were obtained from patients with AML and one patient with CML. Patient samples were collected after informed consent according to institutional guidelines and studies were approved by the Royal Adelaide Hospital and Alfred Hospital Human Ethics Committees. Diagnosis was made using cytomorphology, cytogenetics and leukocyte antigen expression and evaluated according to the French-American-British classification. For patient characteristics see 8) from patients were lysed in a hypotonic buffer  containing 2 mM NaF and Complete Mini EDTA-free protease inhibitor cocktail (Roche). Hypotonic lysis in the absence of detergents was used to ensure that the activity of multi-subunit kinases was preserved during the purification. The lysate was then subjected to centrifugation at 16,000 g for 10 min followed by ultracentrifugation of the supernatant at 186,000 g for 1 h. The clarified lysate was then subjected to fast protein liquid chromatography (FPLC) on a Superdex 200PC 3.2/30 column (Amersham Biosciences). Chromatography was performed using a running buffer  and a flow-rate of 40 µl/min and 40 µl fractions were collected.Primary AML MNCs  and liquid scintillation counting 32P]ATP as substrates as described in Protein kinase activity was examined in (i) aliquots of eluted fractions following chromatography of primary AML samples, (ii) p85 and p110 immunoprecipitates, or (iii) purified recombinant p110 catalytic subunits of PI3K as described in detail in GCAUUGACUAAUCAAAGGATT , AAUAGUGUGAGAAUUUCGCACCACC , and UUACCCAGAUCACCACUAUUAUUUG . Transfection efficiency was monitored using a BLOCK-iT Alexa Fluor red fluorescent oligonucleotide (Invitrogen) and we routinely obtain >85% transfection efficiency using siRNAs HEK 293T cells or primary AML blasts were transfected for 48 h with 100 nM of siRNAs to p110α or a scrambled control using lipofectamine RNAiMAX (1∶300) in OptiMEM medium (Invitrogen) and 0.5% FCS. siRNA sequences for p110α knockdown were Silencer control , Stealth control , TF-1 cells were factor-deprived in RPMI containing 0.5% FCS for 12 h and then stimulated with different GM-CSF concentrations before lysis in NP-40 lysis buffer Cell survival was determined by either trypan blue exclusion, annexin V-FITC (Roche) staining, propidium iodide staining, or counting viable cell number in reference to Flow Count Fluorospheres (BD Biosciences) essentially as described previously Figure S1Inhibition of tyrosine kinase signaling does not affect the survival of AML or CML cells nor the phosphorylation of Ser585 in the GM-CSF and IL-3 βc receptor. (A) MNCs from patients with AML (with AML  were inc(TIF)Click here for additional data file.Figure S2The phosphorylation of Ser585 by the protein kinase activity of PI3K. (A) PI3K was immunoprecipitated from TF-1 cells with antibodies specific for the p110α, p110β and p110δ isoforms of PI3K and then immunoblotted using anti-p85 pAb. Results show that the p110α isoform of PI3K was the most abundant in TF-1 cells. (B) TF-1 cells were lysed in NP40 lysis buffer containing 1% NP40, 10% glycerol, 10 mM Tris-Hcl [pH 7.4], 137 mM NaCl, 10 mM glycerol phosphate, 2 mM Na Vanadate, 2 mM NaFl, 2 mM PMSF, 1 µg/ml leupeptin, 5 µg/ml aprotonin following which PI3K was immunoprecipitated with anti-p85 pAb. Immunoprecipitates were then washed three times in kinase buffer , 0.02% Tween-20) following which 0.25 µCi[γ-32P]ATP, 1 µM non-isotopic ATP and 0.5 µg purified recombinant intra-cytoplasmic domain of βc (βic) were added. Reactions were incubated at 30°C for 30 min following which they were subjected to SDS-PAGE and autoradiography. Mock immunoprecipitates in which no p85 pAb was used as well as no substate (βic) controls were included. LY294002 (10 µM) was added to the kinase reactions where indicated. 32P-labelled p85 and βic are indicated. (C) Constructs for the expression of wild-type p110α (wt), a p110α-4KA mutant  and myc-tagged p85α were transfected into HEK 293T cells. After 48 h, the cells were lysed in NP40 lysis buffer as in (B) and the p85 subunit of PI3K immunoprecipitated with the 9E10 anti-myc mAb. Immunoprecipitates were washed in PI3K kinase buffer  following which 0.25 µCi [γ-32P]ATP, 1 µM non-isotopic ATP and PtdIns/PtdSer were added. Reactions were incubated for 30 min at 30°C following which 32P-PIP were extracted using chloroform/propanol and subject to thin layer chromatography (TLC) as previously described 32P-PIP are indicated. (D) Purified recombinant p110α and p110β (0.5 µg) were incubated with 0.5 µg βic and 0.25 µCi[γ-32P]ATP in a buffer containing 50 mM Hepes [pH 7.4], 5 mM EDTA, 10 mM MgCl2, and 0.25 mM DTT. Where indicated, 10 µM LY294002 was added to the kinase reaction. After 30 min at 30°C, reactions were stopped by the addition of load buffer and subjected to SDS-PAGE. 32P incorporation was detected by autoradiography. Coomassie staining of the gel indicates loading.(TIF)Click here for additional data file.Figure S3The role of PI3K lipid signaling and the regulation of Akt. (A) FDM cells were generated by transduction of mouse E14.5 fetal liver cells with a retrovirus for the expression of HoxB8 in the presence of high concentrations of murine IL-3 as previously described (TIF)Click here for additional data file.Figure S4Inhibition of DNA-PK or ATM kinases does not block Ser585 phosphorylation or the survival of human AML cells. (A) Primary human AML MNCs (AML6) was plated in DNA-PK inhibitor NU7026 (10 µM), PIK-75 (100 nM) or ATM kinase inhibitor CGK733 (10 µM) and cell survival examined at 24 h by annexin V staining and flow cytometry. (B) TF-1 cells were treated with either 10 µM NU7026 or GCK733 for 1 h and then stimulated with the indicated concentrations of GM-CSF for 20 min. Cells were then lysed and βc immunoprecipitates were blotted with indicated antibodies.(TIF)Click here for additional data file.Figure S5siRNA-mediated knockdown of p110α results in down-regulation of Ser585 phosphorylation. (A) Primary human AML MNCs (AML10–14) were transduced with 100 nM control or siRNA-p110α-1 (Ambion) for the down-regulation of the p110α catalytic subunit of PI3K. After 48 h, cells were lysed and the βc subunit of the GM-CSF/IL-3 receptor immunoprecipitated followed by Western blotting with the indicated antibodies. Phospho-βcSer585 signals were quantified by laser densitometry. Relative intensity (%) of quantified signals are indicated under the immunoblots. (B) Primary human AML MNCs (AML6 and AML9) were plated in PIK-75 (100 nM), TGX-221 (1 µM), IC87114 (5 µM), or AS25424 (100 nM) and cell survival examined at 24 h.(TIF)Click here for additional data file.Table S1Selectivity of PI3K inhibitors.(DOC)Click here for additional data file.Table S2Primary human AML samples used and clinical details. AML samples used (AML1–AML15) were obtained from apheresis product, BM, or peripheral blood samples. Patient samples were collected after informed consent according to institutional guidelines and studies were approved by the Royal Adelaide Hospital Human Ethics Committee and Alfred Hospital Human Ethics Committee. +, white cell count (WCC) (×109/l); ♦, normal karyotype (NK). Complex indicates at least three abnormalities.(DOC)Click here for additional data file.