Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides.In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin.Here, we studied the role of Ugd phosphorylation. We first confirmed Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology. In bacteria, protein phosphorylation is catalyzed mainly by histidine-kinases which are key enzymes of the so-called “two component systems” Escherichia coli produces two BY-kinases, Wzc and Etk. They are respectively encoded by genes located at approximately 46 min and 22 min on the E. coli chromosome in two gene-clusters both involved in the biosynthesis of extracellular polysaccharides E. coli, a UDP-glucose dehydrogenase, is one of the first identified substrates of a BY-kinase in vitro by the BY-kinase Wzc. Other types of sugar-dehydrogenases, including the UDP-glucose dehydrogenase of Bacillus subtilis UgdBsStaphylococcus aureusE. coliE. coli. In addition, UDPGA participates in the production of a sugar derivative, UDP-4-amino-4-deoxy-L-arabinose (L-Ara4N) which is a crucial element in bacterial resistance to antibiotics such as polymyxin and cationic peptides of the innate immune system E. coli K-12, we had previously demonstrated that Wzc autophosphorylation influences the production of colanic acid, etk knock-out mutant is much less resistant to polymyxin than a wild-type E. coli K-12 strain. Protein Ugd of in vivo evidence that, depending on the growth conditions, Ugd can be phosphorylated by either Wzc or Etk. We show that Ugd is phosphorylated on the same site by both BY-kinases, Wzc and Etk. We further show that Wzc-mediated phosphorylation of Ugd specifically affects the biosynthesis of colanic acid whereas resistance to the cationic antimicrobial peptide polymyxin is dependent upon Ugd phosphorylation by Etk. These data represent the first report of a bacterial protein phosphorylated by two distinct tyrosine-kinases. They contribute to define the role of tyrosine phosphorylation in bacteria and provide a basis for an emerging regulatory network in E.coli.In this work, we provide in vitro by the catalytic domain of Wzc, Wzccytoin vivo phosphorylation of Ugd by Wzc, we used a strain that encodes the transcriptional regulator RcsA wzc and ugdetk, which is not expressed in our culture conditions wzc knock-out mutant ugd gene was inactivated . To assess if Etk can phosphorylate Ugd, we also analyzed total protein extracts of wild-type, ugd-deficient and etk-deficient strains of E. coli by immunorevelation with the Ugd-specific antibody and the anti-phosphotyrosine antibody   . We hypocyto (kinase/substrate ratio of 1/100), we observed that Ugd was phosphorylated whereas no radioactive signal was detected for UgdY71F (SR = 62+/−3 Å) confirming that the overall structure of Ugd was most likely not affected by the Tyr to Phe mutation. To confirm Tyr71 as the phosphorylation site responsible for activating the enzyme, the dehydrogenase activity of Ugd and UgdY71F was measured with or without prior incubation with Wzccyto and ATP  with an episomal copy of either the ugd gene (Δugd+ugd strain) or the ugd Y71F allele (Δugd+ugdY71F strain)  . Total p strain) . We obseE. coli K-12 producing colanic acid exhibits a mucoid phenotype, and on plates, colonies have a fatty and shiny appearance. In contrast, smaller and duller colonies are observed when colanic acid is not produced. Therefore, we compared the colony morphology of strains producing Ugd, wild-type form or mutated on Tyr71 . These data nevertheless confirm the two distinct phenotypes: mucoid for the wild-type and Δugd+ugd strains, and non-mucoid for the Δugd and Δugd+ugdY71F strains. They also confirm that phosphorylation of UgdTyr71 by Wzc affects colanic acid production.on Tyr71 . We firsnic acid . By contd strain . These dd strain . Quantif UgdY71F . AccordiE. coli K-12, Ugd catalyzes the first step of the biosynthesis of L-Ara4N, which confers bacterial resistance to cationic antimicrobial peptides and antibiotics such as polymyxin E. coli resistance to polymyxin depends also on the expression of etkin vivo by Etk when E. coli was grown under culture conditions allowing resistance to polymyxin in vivo phosphorylation of Ugd would also occur on Tyr71. For this, the Δugd+ugd and the Δugd+ugdY71F strains were grown in the conditions allowing polymyxin resistance and total protein extracts were prepared and analyzed by SDS-PAGE and immunoblotting. As shown in ugd+ugd and Δugd+ugdY71F strains. However, when immunoblotted with the anti-phosphotyrosine antibody, a phosphorylation signal was detected only when the wild-type form of Ugd was produced. This observation confirmed that Ugd was specifically phosphorylated in vivo on Tyr71 by Etk and that Ugd Tyr71 phosphorylation could consequently influence polymyxin resistance.In E. coli strains were grown in the culture medium allowing resistance to polymyxin, incubated for 1 hour in the presence of varying concentrations of polymyxin and plated onto agar-LB plates. Survival rates are expressed as the percentage of the number of colonies formed with a strain grown in the presence of polymyxin with respect to the number of colonies obtained with the same strain grown in the absence of the antibiotic. Optimal resistance of the wild-type strain was 39% or 25% survival in the presence of 2.5 or 5 µg/ml polymyxin, respectively. As expected, the Δugd strain exhibited 7% to 1% survival, which represents only 17% to 3% of the wild-type strain resistance , which includes 19 genes, contains a stem-loop transcriptional attenuator which is located immediately after the third gene, namely wzcwzc might therefore be expressed independently of the other cps genes and be involved in the phosphorylation of other proteins so as to regulate other cellular functions. There are also arguments for the second hypothesis, namely that other tyrosine kinases could be encoded by the genome of E. coli. Several proteins of unknown function harbor the Walker A and B motifs E. coli but also in a large number of proteobacteria and firmicutes in which BY-kinases have been identified In this work, we showed that Ugd of -kinases . In E. cStreptococcus thermophilus, by the BY-kinase EpsD We established that Wzc-mediated phosphorylation of Ugd influences the production of colanic acid . It has E. coli K-12, the phosphorylation of Ugd could also have side effects on the production of other polysaccharidic compounds. A recent study has demonstrated that variation of polysaccharide chain length depends on the UDP-glucose (UDPG) concentration available in the bacterial cell E.coliE. coli K-12, forming thus a novel LPS glycoform henceforth called MLPSLPS could also be affected by phosphorylation of Ugd . Antibiotics were added at the following concentrations: 50 µg/ml ampicillin, 25 µg/ml kanamycin, 15 µg/ml tetracyclin.Strains and plasmids used in this study are listed in ugd in E. coli W3110 or JM83 strain was performed by one-step inactivation as previously described ugd was replaced with the kanamycin resistance cassette (KanR)  was cloned into the pQE30 vector previously opened with BamHI and HindIII. Constructs were checked by DNA sequencing. Primers used in this study are described in Gene replacement of e (KanR) . Two-wayE.coli cells collected in the postexponential phase of growth. RNA was purified with the High Pure RNA isolation kit (Roche). Contaminating DNA was removed by additional treatment for 20 min at 37°C with 10 U DNase I (Roche). RT-PCR amplification was carried out with the SuperScript II reverse Transcriptase (Invitrogen) following the manufacturer's recommendations. A control sample without reverse transcriptase was included to confirm the absence of contaminating DNA.Total RNA was prepared from cyto, Etkcyto, Ugd or UgdY71F were expressed in E. coli XL1-Blue cells. The purification procedure and in vitro kinase assays were performed as previously described cyto ranging from 0.002 µg to 1 µg. The reaction mixtures were then analyzed by SDS-PAGE. After electrophoresis, gels were soaked in 20% TCA for 10 min at 90°C, stained with Coomassie blue and dried. Radioactive proteins were visualized by autoradiography using direct exposure films. Ugd dehydrogenase activity measurement were performed in a thermostated cuvette at 37 °C, on a PowerWave 340 BIO-TEK spectrophotometer as described Wzc2PO4 pH 7.8, 300 mM NaCl, 10% glycerol and 150 mM imidazol with a Zetasizer Nano series Malvern instrument. A light path of 3 mm was used. Protein concentration was of 14 mg/mL and 8 mg/mL for Ugd and UgdY71F, respectively.DLS measurements were performed at 18 °C in a buffer containing 50 mM NaHBacterial cell extracts were analyzed by Western blotting after SDS-PAGE. Proteins Wzc and Ugd were detected using specific monoclonal antibodies prepared in our laboratory according to the procedure described The method used was based on the procedure previously described 2SO4/H2O (6∶1 v/v). The mixture was prepared at room temperature, then heated at 100 °C for 20 min, and finally cooled down to room temperature. For each sample, absorbance at 396 nm and 427 nm was measured either directly (control sample (A-co)) or after addition of 100 µl of cysteine hydrochloride (cysteine sample (A-cy)). Indeed, biological extracts often contain compounds which under heating with H2SO4 yield brown products absorbing between 396 and 427 nm. The absorption due to this unspecific reaction was subtracted from the total absorption of the sample: A396-co and A427-co were respectively subtracted from A396-cy and A427-cy to obtain DA396 and DA427. Values of (DA396–DA427) were directly correlated to methylpentose concentration by using a standard curve obtained with a fucose concentration ranging from 5 to 100 mg/ml.Quantification of colanic acid was carried out according to 2+. Cultures were then harvested and washed three times with a N-minimal medium at pH 5.8 (mild acid conditions), in the presence of 0.2% glucose and 10 µM Mg2+ (low magnesium conditions) and 1 µM Fe3+ (Resistance-Inducing medium) Tested strains were grown for 12 h in LB medium at 37°C, diluted 100 fold and grown again overnight in N-minimal medium at pH 7.7, in the presence of 0.2% glucose and 10 mM MgFigure S1Analysis of the Ugd amino acids sequence to characterize the phosphorylated tyrosine. (A) Comparison of both the amino-acid sequences and the predicted secondary structure of Ugd and YwqF. β, α, and η indicate β-sheet, α-helices and 3.10 helices, respectively. Secondary structure elements of Ugd and YwqF have been predicted using Streptococcus pyogenes UDP-glucose dehydrogenases (PDB code 1DL1) and Pseudomonas aeruginosa GDP-mannose dehydrogenase (PDB code 1MV8) as templates respectively  Conserved tyrosines are indicated in cyan. Tyr70 of YwqF and Tyr71 of Ugd are highlighted in green. (B) Autoradiography of SDS-PAGE on which reaction mixtures containing [γ-32P]ATP and either Ugd and Wzccyto (lane 1), or UgdY10F and Wzccyto (lane 2), or UgdY150F and Wzccyto (lane 3), or UgdY249F and Wzccyto (lane 4), or UgdY335F and Wzccyto (lane 5), or UgdY380F and Wzccyto (lane 6) were analyzed. References 1.Gouet, P., Robert, X., and Courcelle, E. (2003) ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31: 3320-3323. 2.Rost, B., and Liu, J. (2003) The PredictProtein server. Nucleic Acids Res 31: 3300-3304.(21.88 MB TIF)Click here for additional data file.Table S1Bacterial strains and plasmids used in this study(0.04 MB DOC)Click here for additional data file.Table S2Primers used in this study(0.04 MB DOC)Click here for additional data file.


Anandamide-dependent protection was reduced by pretreatment with LY294002  and unaffected by U0126 (inhibitor of extracellularly-regulated kinase). Interestingly, phosphorylation of c-Jun-NH2-terminal kinase (JNK) in cells exposed to 6-OHDA was strongly reduced by anandamide pre-treatment. Furthermore, 6-OHDA induced c-Jun activation and increased Bim expression, both of which were inhibited by anandamide. Together, these data demonstrate antiapoptotic effects of anandamide and also suggest a role for activation of PI3K and inhibition of JNK signalling in anandamide-mediated protection against 6-OHDA.6-hydroxydopamine (6-OHDA) is a selective neurotoxin that is widely used to investigate cell death and protective strategies in models of Parkinson's disease. Here, we investigated the effects of the endogenous cannabinoid, anandamide, on 6-OHDA-induced toxicity in rat adrenal phaeochromocytoma PC12 cells. Morphological analysis and caspase-3 activity assay revealed that anandamide inhibited 6-OHDA-induced apoptosis. The protection was not affected by antagonists of either cannabinoid receptors (CB Fluorescent units were converted to nanomoles of AMC released using a standard curve generated with free AMC and subsequently related to protein concentration.The activity of caspase-3-like enzymes (DEVDases) was determined fluorometrically as reported previously  with som2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.5 mM DTT, 0.1% phenylmethylsulphonyl fluoride (PMSF), 1% aprotinin, 5 mM NaF, and 1 mM Na3VO4). Cellular debris was spun down at 20,000 × g for 1 minute and supernatant was taken to determine the protein content using Bradford reagent with bovine serum albumin (BSA) as the standard.Following experimental treatments cells were scraped from the culture flasks and centrifuged at 150 × g for 5 minutes at 4°C. After washing in PBS cells were lysed using whole cell lysis buffer , 5%  β-mercaptoethanol, 4% glycerol, 1 mM PMSF, 0.01% bromophenol blue) and boiled at 95°C for 5 minutes. Proteins were separated by 10%–15% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. Membranes were blocked for 1 hour in PBS containing 0.05% Tween 20 and 5% (w/v) non-fat dried milk. Membranes were probed with antibodies (1  :  1000) overnight at 4°C followed by appropriate horseradish peroxidise-conjugated goat secondary antibody at 1  :  10,000  for 2 hours at room temperature. Protein bands were visualized using Supersignal West pico system (Pierce).40 post hoc test, for which levels of P < .05 were considered to be significant.Values are expressed as means ± SEM of 3 separate experiments unless otherwise indicated. Statistical analysis was performed using repeated-measures ANOVA followed by Bonferroni multiple comparisons μM anandamide for 24 hour followed by exposure to 100 μM 6-OHDA for a further 24 hours. Visualisation of nuclear morphology by DAPI staining demonstrated that pre-treatment of PC12 cells with anandamide inhibited 6-OHDA-induced apoptosis  was examined. PC12 cells were treated with 0–50 andamide . This waandamide . These d1 receptor  also did not reverse the protection /Akt and mitogen-activated protein kinase (MAPK) signalling pathways, both of which are linked to prosurvival signalling –32. To gtoxicity . Pre-treμM anandamide resulted in a transient increase in ERK1/2 phosphorylation at 6 hours that returned to basal levels by 24 hours . This inhibitor reduced phosphorylation of c-Jun by 6-OHDA and also caused a delay and a reduction in cleavage of caspase-3 to its active p17 fragment . Anandaiewed by  or nontoiewed by  to PC12 iewed by , 41, 43,iewed by . For exaiewed by . Furtherdopamine . Althoug1/CB2 or TRPV1 receptors and required a breakdown of anandamide to ethanolamine [A possible role of metabolites of anandamide in the regulation of PC12 cell fate cannot be ruled out. Anandamide is enzymatically hydrolysed to ethanolamine and arachidonic acid . In factnolamine . 2O2-induced death, suggesting that anandamide does not inhibit oxidative stress due to 6-OHDA. However, H2O2 is not the only product of 6-OHDA auto-oxidation. In addition, superoxide anions and p-quinone, may contribute to reactive oxygen species (ROS) production [It has been reported that induction of oxidative stress is involved in 6-OHDA-induced toxicity and antioxidants can protect against hydrogen peroxide and superoxide anion-mediated cell death , 45–47. oduction –50 and mEL. Cannabinoids have been shown to activate PI3K/Akt and MEK/ERK signalling pathways , 52. WhiEL. Furthermore, the JNK inhibitor SP600125 caused a partial reduction in caspase-3 cleavage due to 6-OHDA. This was in contrast to complete inhibition by anandamide. These findings are in agreement with a previous report showing that SP600125 partially protects against 6-OHDA toxicity in PC12 cells [JNKs are members of the mitogen-activated protein kinase (MAPK) pathway that is activated in response to many extracellular stimuli and different forms of environmental stress . Our fin12 cells . Taken tBim [EL, Bmf, and Bcl-2 [EL levels and function are regulated by phosphorylation by ERK, JNK and possibly Akt [EL at Ser65 by active JNK potentiates its pro-apoptotic activity [EL. However, since the level of cytoprotection by anandamide was far greater than that due to SP600125, despite similar JNK inhibition, it strongly suggests that anandamide utilises additional mechanisms to inhibit 6-OHDA toxicity. JNK promotes apoptosis in a number of ways. Through phosphorylation and activation of c-Jun it stimulates the transcription of c-Jun target genes, including Bim . In addind Bcl-2 , 53. Foribly Akt , 55. Phoactivity . These dIt should be considered that in contrast to the data supporting endocannabinoid-mediated JNK inhibition, there are data reported by others –59 indicHere, for the first time we demonstrate that anandamide protects PC12 cells against 6-OHDA-induced apoptosis. These data and those of other studies may suggest potential therapeutic benefits of elevating anandamide and possibly other endocannabinoids in preventing the degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease.


The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease. Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that  delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression. Cells rely on the mammalian target of rapamycin kinase (mTOR) to sense growth factors. Inhibition of all forms of mTOR using newly developed inhibitors of its active site reveals new insights into the function of two mTOR-containing protein complexes and their potential as therapeutic targets. The mammalian target of rapamycin (mTOR) is a serine-threonine kinase related to the lipid kinases of the phosphoinositide 3-kinase (PI3K) family. mTOR exists in two complexes, mTORC1  and mTORmTORC2 is insensitive to rapamycin, and selective inhibitors of this complex have not been described. Partly because acute pharmacological inhibition of mTORC2 has not been possible, the functions of mTORC2 are less well understood than those of mTORC1. mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt ,6 and SGThe disruption of mTORC2 by different genetic and pharmacological approaches has variable effects on Akt phosphorylation. Targeting mTORC2 by RNA interference (RNAi) ,14, homoSeveral small molecules have been identified that directly inhibit mTOR by targeting the ATP binding site; these include LY294002, PI-103, and NVP-BEZ235 –22. ThesWe recently reported the synthesis of pyrazolopyrimidines that inhibit members of the PI3K family, including mTOR . Two of Another small-molecule ATP-competitive mTOR inhibitor called Torin1 was reported while our manuscript was in the process of publication .50 values) of 8 nM and 80 nM, respectively. As expected for active-site inhibitors, PP242 and PP30 inhibit mTOR in both mTORC1 and mTORC2 —were inhibited more than 80% [50 values for PP242 against these kinases in vitro using purified proteins. In these assays, PP242 was relatively inactive against PKC-beta, RET, or JAK2 but inhibited PKC-alpha with an in vitro IC50 of 50 nM  status in these two conditions  at T389 and the eIF4E-binding-protein, 4EBP1, at multiple sites. Our proliferation experiments suggest that rapamycin and PP242 have distinct effects on mTORC1. We compared the effects of acute treatment with rapamycin and PP242 on S6K, ribosomal protein S6 (S6), and 4EBP1 phosphorylation A to see −/− MEFs that lack mTORC2 , allowing proteins bound to eIF4E to be examined. Rapamycin caused partial inhibition of the insulin-stimulated release of 4EPB1 from eIF4E , suggesting that downstream mTORC1 substrates might be responsible for PP242′s strong anti-proliferative effects. Interestingly, we observe that phosphorylation of the mTORC1 substrate 4EBP1 is partially resistant to rapamycin treatment at concentrations that fully inhibit S6K, whereas PP242 completely inhibits both S6K and 4EBP1. Because rapamycin can only partially inhibit the phosphorylation of 4EBP1, but it can fully in inhibit the phosphorylation of S6K, rapamycin appears to be a substrate-selective inhibitor of mTORC1. Consistent with this finding, experiments with purified proteins have shown that rapamycin/FKBP12 only partially inhibits the in vitro phosphorylation of 4EBP1 at Ser 65 by mTOR but can fully inhibit the in vitro phosphorylation of S6K [−/− MEFs is more sensitive to PP242 than rapamycin , Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail, and 2 mM PMSF. For 2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail without EDTA and 2 mM PMSF).Except where indicated otherwise, cells were serum starved overnight and incubated with inhibitors or 0.1% DMSO for 30 min prior to stimulation with 100 nM insulin for 10 min. All inhibitors were either synthesized as previously described ,24,55 orL6 myotubes from one well of a six-well plate were lysed in 300 μl of cap lysis buffer as described above. 50 μl of detergent-free cap lysis buffer and 20 μl of pre-washed cap beads were added to 150 μl of cleared lysate and incubated at 4 °C overnight with tumbling. The beads were washed twice with 400 μl of cap wash buffer (cap lysis buffer with 0.5% NP-40 instead of 1% NP-40) and twice with 500 μl of PBS. The beads were boiled in SDS-PAGE sample buffer and the retained proteins analyzed by Western blot. All antibodies were from Cell Signaling Technologies except for the anti-eIF4E antibody, which was from BD Biosciences.2, 5 mM ß-glycerol phosphate, 0.05 mg/ml phosphatidylserine, 0.03% Triton X-100, 0.5 mg/ml BSA, 2.5 mM DTT, 100 μM CaCl2, 1 μM PMA, 10 μM ATP, and 15 μCi/ml of γ-32P-ATP. Inhibitors were tested in a four-fold dilution series from 10 μM to 600 pM, and four measurements were made at each concentration. The kinase reaction was terminated by spotting onto nitrocellulose, which was washed 5 times with 1 M NaCl/1% phosphoric acid. The radioactivity remaining on the nitrocellulose sheet was quantified by phosphorimaging, and IC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package.Phosphorylation of histone H1 (4 μM) by PKC was assayed in a buffer containing 200 ng/ml recombinant kinase, 25 mM HEPES pH 7.5, 10 mM MgClPDK1, mTORC1, and mTORC2 were assayed as previously described .++ and Mg++. The cells were washed three times with PBS and the blocked and permeabilized with 5% goat serum in PBS with 0.3% Triton X-100 (PBS-GS-TX). Primary antibodies to S473  and T308  were added at 1:1000 and 1:500, respectively, in PBS-GS-TX, and the plates were incubated at 4 °C overnight. The plates were then washed three times with PBS, and goat anti-rabbit secondary antibody (Pierce Biotechnology) was added at 0.01 μg/ml in PBS-GS-TX. After 1 h at room temperature, plates were washed three times with PBS. ELISA chemiluminescent reagent  was added to each well and after 1 min, the plate was read in a luminescence plate reader using a 100-ms integration time. The pAkt signal from pT308 and pS473 was normalized to control wells, so that 0 represents the level of pAkt in serum starved cells and 1 represents the level upon insulin stimulation. EC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. The significance of differences between EC50 values was evaluated using the F test.L6 myotubes were grown and differentiated in 96-well plates. The outside wells of the plate were not used for the experiment, but were kept filled with media. Following stimulation, cells were fixed for 15 min with 4% formaldehyde in PBS with CaAkt was transfected into HEK293 cells using Lipofectamine 2000 according the manufacturers protocol. Two days after transfection, cells were serum starved overnight and the next day they were treated with inhibitors and processed for western blotting as described above.NIH 3T3 cells were plated on poly-lysine coated coverslips at 30% confluence the day before the experiment. Following treatment with PP242 or 0.1% DMSO for 8 h in 10% serum growth medium, the actin cytoskeleton was stained as previously described [Primary MEFs were transfected with a bicistronic reporter  containi35S35S (NEN) was added to each well and the cells were labeled for 4 h. Cells were washed once with ice-cold PBS, and lysed as described above for Western blotting. Following separation by SDS-PAGE, and transfer to nitrocellulose, 35S-labeled proteins were visualized by autoradiography with film. For quantitation, the membrane was exposed to a phosphorimager screen and the resulting image was quantified in ImageJ. Differences in 35S incorporation were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package.Primary MEFs grown to 70% confluence in six-well plates were incubated overnight in either 10% Serum (Steady State), kinase inhibitors in 10% serum, or 0.1% serum (starved). Cells were then washed once with DMEM lacking cysteine and methionine (DMEM-noS), and the medium was replaced with DMEM-noS including dialyzed serum and kinase inhibitors as indicated. After incubation for 1 h, 50 μCi of Expre2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 μl of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer (Fisher PowerGen 125 with Omni-Tip probe). Protein concentration of the cleared lysate was measured by Bradford assay and 5–10 μg of protein was analyzed by Western blot as described above.Drugs were prepared in 100 μl of vehicle containing 20% DMSO, 40% PEG-400, and 40% saline. Six-wk-old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242 (0.4 mg), rapamycin (0.1 mg), or vehicle alone was injected IP. After 30 min for the rapamycin-treated mouse or 10 min for the PP242 and vehicle-treated mice, 250 mU of insulin in 100 μl of saline was injected IP. 15 min after the insulin injection, the mice were killed by CO−/− MEFs were plated in 96-well plates at approximately 30% confluence and left overnight to adhere. The following day cells were treated with PP242, rapamycin, or vehicle (0.1% DMSO). After 72 h of treatment, 10 μl of 440 μM resazurin sodium salt (Sigma) was added to each well, and after 18 h, the florescence intensity in each well was measured using a top-reading florescent plate reader with excitation at 530 nm and emission at 590 nm.Wild-type and SIN1Figure S1L6 myotubes were pre-treated with PP242 or DMSO for 30 min and stimulated with insulin for the indicated times prior to lysis and analysis by Western blotting.(4.31 MB AI).Click here for additional data file.Figure S2(A) 4EBP1 phosphorylation at T70 is not inhibited by either PP242 or rapamycin. L6 myotube lysates from −/− and wild-type (WT) MEFs. Western blotting from (B) 4EBP1 phosphorylation is inhibited by PP242 with similar potency in SIN1(4.97 MB AI).Click here for additional data file.Figure S3L6 myotube lysates from (2.44 MB AI).Click here for additional data file.Figure S4(A) Renilla (cap-dependent) luciferase activity from samples in p = 0.4, ANOVA).(B) Firefly (IRES-dependent) luciferase activity from samples in (1.55 MB AI).Click here for additional data file.Table S1(19 KB DOC)Click here for additional data file.


Environmental monitoring for pharmaceuticals and endocrine disruptors in the aquatic environment traditionally employs a variety of methods including analytical chemistry, as well as a variety of histological and biochemical endpoints that correlate with the fish fitness. It is now clear that analytical chemistry alone is insufficient to identify aquatic environments that are compromised because these measurements do not identify the biologically available dose. The biological endpoints that are measured are important because they relate to known impairments; however, they are not specific to the contaminants and often focus on only a few known endpoints. These studies can be enhanced by looking more broadly at changes in gene expression, especially if the analysis focuses on biochemical pathways. The present study was designed to obtain additional information for well-characterized sites adjacent to sewage treatment plants in MN that are thought to be impacted by endocrine disruptors.Pimephales promelas) that have been caged for 48 h in each of the aquatic environments. We find that the gene expression changes are characteristic and unique at each of the five sites. Also, fish exposed to two of the sites, 7 and 12, present a more aggressive behavior compared to control fish.Here we examine five sites that have been previously characterized and examine changes in gene expression in fathead minnows (Our results show that a short-term exposure to sewage treatment plant effluents was able to induce a site-specific gene expression pattern in the fathead minnow gonad and liver. The short-term exposure was also enough to affect fish sexual behavior. Our results also show that microarray analysis can be very useful at determining potential exposure to chemicals, and could be used routinely as a tool for environmental monitoring. Effluents from sewage treatment plants are a big source of endocrine-disrupting compounds (EDCs) into the aquatic environment. Exposure of aquatic organisms to EDCs has been linked to adverse physiological effects such as reduced fertility , interseLee et al. ,7 analyzIn the Lee study , female These studies indicated the presence of EDCs by analyzing biological characteristics, such as hormone and vitellogenin levels, in the common carp. The studies identified sewage treatment plant effluents as a potential source of EDCs. Additionally, fish located at sites upstream of sewage treatment plant effluent draining agricultural and forested land showed indications of EDCs. The present study was designed to obtain additional information using genomics for some of these well-characterized sites in MN that are thought to be impacted by EDCs.Microarray analysis has developed in recent years to the point where it can be used to gain mechanistic understanding about how fish health is impacted by contaminants. This method provides an unbiased open assessment of the health of fish exposed to polluted aquatic environments. A few studies have used microarrays to analyze the gene expression signature in fish exposed to polluted field sites ,9. If th2) and estrone (E1), and the pharmaceutical estrogen 17α-ethinylestradiol (EE2), found in birth control pills, have been identified [2 and zearalenone) and progestins (melengestrol acetate) [A variety of anthropogenic chemicals such as industrial chemicals, surfactants and pesticides are known to have some estrogenic potency ,10,11. Bentified . It is aentified , estrogeacetate) . MinnesoPimephales promelas – FHM) that were caged for 48 h in the different aquatic environments. We also examined whether 48 h exposure to water from the five sites of interest affected the ability of males to compete for nests and females. For many fish, including the FHM, acquisition of spawning territory is a competitive process in which more aggressive males acquire and maintain spawning territories , whereas subordinate males often fail to reproduce. Past studies indicated that exposure to environmental estrogens can lead to reduced ability of males to acquire nests, whereas exposure to androgens can increase the performance of aggressive behaviors and enhance nest acquisition [Here we examine five sites that have been previously characterized by Lee et al. ,7 by exauisition .We hypothesized that fish exposed to water downstream from sewage treatment plants will have a different gene expression signature than those exposed to upstream waters, probably showing a more estrogenic signature, as waters downstream of sewage treatment plants have been shown to be estrogenic in some cases ,16. For Cyprinus carpio). The five sites were the following: Site 7  is located on Shell Rock River near Albert, Lea, MN. Site 11  is on the Des Moines River, upstream of Windom. Site 12  is on the Des Moines River, downstream of Windom and down stream from a sewage treatment facility. Site 13  is located on Rock River upstream of Luverne, MN. Although the high percentage of agricultural land and the number of animal feedlots in the drainage basin provides the potential for runoff of animal wastes to the streams, those characteristics do not guarantee that runoff into the stream has occurred or was present when the fish were exposed. A fifth site, Site 21 at Jewitt's Creek  downstream of Litchfield, MN, was identified in Lee et al. [Five sites were selected for exposing FHM to possible EDCs in the natural environment Figure . These sA 22,000 gene microarray specific for FHM  was usedp-value < 0.05) in relation to their fold-expression compared to Site 11  groups at each field site. We were able to identify human homologs for about 50% of the differentially expressed genes.http://david.abcc.ncifcrf.gov, [We used the functional clustering implemented in the web-based application DAVID  to dThe behavioral test found no effects on behavior in fish exposed at site 11, which lends support to the gene expression analyses that fish from the site were the least affected. Fish from site 13 also presented unchanged behavior. The fish from site 21 showed initial increases in aggressive behavior, but were not able to maintain possession of the nests; by the third day of behavioral experiments there were no significant differences in nest-holding ability between exposed and control males. Fish exposed to Sites 7 and 12 consistently dominated control fish and actively defended the majority of the nests over the course of the whole experiment. Figure These studies were designed to re-examine specific locations in rivers in Minnesota that had previously been studied by Lee et al. ,7. The LThe expression fingerprints for fish exposed to five different field locations showed unique patterns for fish within each location that differed among the five regions picked  for both the liver and the gonad. Fish exposed at the same site showed similar gene expression profiles. Sites 11 and 12 were closely related consistent with their locations on the same river, up-stream and down-stream respectively, from a sewage treatment facility.Site 11 male carp had the fewest biological anomalies; presenting only low GSI . FHM exp2 levels were slightly higher (250 pg/mL compared to 150 pg/mL). In the present study, responses to site 7 appeared very complex, since we had from 650–800 genes altered by the treatment, but distinguishable from other sites by its molecular fingerprints. We did not find Vtg mRNA up-regulated after 48 h. We expect this site would be highly variable as well because it not only receives effluent from the sewage treatment facility but also because it gets a lot of agricultural runoff.Site 7 is located on the Shell Rock River down stream from the WWTP near Albert Lea, MN. Male carp at this location [Males normally have a balance between estrogen and androgen hormones. Both types of hormones play a very important role in homeostasis. In site 7, we found several affected genes  that could be decreasing the effects of estrogenic compounds, thereby tipping the hormonal balance towards more androgenic pathways and resulting in more aggressive behavior. Prohibitin and prohibitin 2 were up-regulated both in the gonad and liver of fish exposed to site 7 waters. Prohibitin, a potential tumor suppressor, has been shown to function as a potent transcriptional corepressor for estrogen receptor alpha (ERα) . The rep2 and Vtg levels and low plasma 11-KT levels, suggesting impairment in steroidogenesis and potential exposure to estrogenic contaminants [Site 13, located upstream from Luverne, MN, has a considerable number of animal feeding operations within 5 miles of the site. Male carp at this location presented with high plasma Eaminants . They alaminants , suggestaminants . There iaminants . RORα upaminants .Xenopus laevis [2 in the gonad of FHM [Finally site 21, located on Jewitt's Creek near Litchfield, was chosen based on a separate study by Lee et al. . It was s laevis ,27. Signs laevis  and apops laevis  among ots laevis  and it hd of FHM . This coThe fish from Sites 7 and 12 were more successful at acquiring nests than control males, while fish from Site 13 showed no significant changes in behavior. The observed increases in the ability of males from sites 7 and 12 to occupy and defend nests are consistent with findings of others who found that waterborne androgens increased aggressive behavior and nest acquisition . The fisSites 13 and 21 presented up-regulation of several thyroid hormone associated proteins (TRAP), both in the gonad (TRAP5 in both sites and TRAP230 in site 13) and the liver (TRAP3 in both sites and TRAP4 in site 21). The TRAP complex is a coactivator complex that interacts and modulates the activity of thyroid hormone receptors ,33, suggCYP20A1 is up-regulated in these sites although that is not the case of some of the other CYPs involved in the metabolism of xenobiotics compounds, like CYPA1. The lack of CYP1A induction could also be related to the presence of flame retardants, as some have been shown to be antagonists that can reduce the induction of CYP1A by a more potent agonist ,38.Ornithine decarboxylase antizyme 2, an ornithine decarboxylase (ODC) inhibitor , was up-In order to elucidate the causes of the increased nest acquisition rates (these are mediated by aggressive behavior), we compared the differentially expressed genes from sites 7 and 12. We expected these changes to be related to androgen exposure, as circulating androgens have been related to aggressive behavior in fish ,44. ThirCAV1, a membrane protein up-regulated at sites 7 and 12, is one of the few proteins known to bind cholesterol, the precursor of steroid hormones, tightly and specifically. It has a very important role in cholesterol homeostasis. Caveolins have also been linked to reverse cholesterol transport, where excess free cholesterol is released into the plasma . CAV1 haOur results show that even a short exposure (48 h) to streams adjacent to sewage treatment plants was able to induce a site-specific gene expression pattern in the fathead minnow gonads and livers. The short-term exposure was also enough to affect the fish sexual behavior at two of the sites. These findings suggest that gene array analysis can complement chemical analysis as a monitoring tool. These findings also suggest that microarray analysis is relatively robust especially when used in conjunction with other more established methods to define contaminated aquatic environments for risk assessment and environmental monitoring.To conduct the field exposures at each field site, FHM were transported from the laboratory to the field site in aerated, insulated tanks. At each site, 25 males and 25 females were placed in separate wire mesh minnow traps with the entrance funnel plugged. The traps were anchored to the bottom in the stream current with the top of the traps submerged. Fish were removed from the traps 48 h later and transported back to the laboratory in aerated, insulated tanks containing the stream water. Immediately upon arrival at the laboratory, four males and four females were sacrificed as described for the laboratory exposures. Liver and gonads were removed and stored in liquid nitrogen until processed for arrays.All procedures involving live fish were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).free  following the manufacturer's protocol. The quality of total RNA was assessed with the Agilent 2100 BioAnalyzer  and the quantity was determined on a NanoDrop spectrophotometer . RNA was stored at -80°C until further use.Total RNA was isolated from gonadal tissue with the RNA Stat-60 reagent  as described previously . RNA pelhttp://www.ncbi.nlm.nih.gov/geo/; Accession series record number GSE16645).Fathead minnow 22,000 gene arrays were designed by EcoArray  and were purchased from Agilent. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control female and male tissues . Four replicates, each consisting of a different individual, were analyzed for each of the treatment sites . cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer's kits and protocols . Gonad and liver samples from the fish at the sites were labeled with Cy5 while the reference sample was labeled with Cy3. Consistent with the Minimum Information about a Microarray Experiment (MIAME) standards , text ve2 transformed signal ratio between the experimental channel and the reference channel was calculated for each spot, followed by within-array lowess transformation and between array scale normalization on median intensities [Microarray image processing and data pre-processing were performed using Agilent's Feature Extraction software v 9.5 . The intensity of each spot was summarized by the median pixel intensity. A logensities .2 transformed signal ratios of each probe individually, followed by Tukey-HSD pair-wise comparisons to determine genes whose expression was significantly regulated between sites. Statistical significance was determined at a p-value of < 0.05 with an FDR threshold of 16%. FDR was calculated using Benjamini-Hochberg approach [One-way ANOVA was performed on normalized logapproach . After tIndividual fish were transferred into 10-L glass test aquaria for behavioral testing, each of which was supplied with clean well water (50 ml/min). Prior to placement in tanks, both site water-exposed and control males were acclimated to the temperature of the observation tank (25°C) over a 4-h period. For each one of the sites, we tested 7–10 behavioral replicates. Each one of these replicates consisted of a control male added along with the site water-exposed male to an aquaria containing one unexposed female and a nest. Male fish were selected so that their total lengths were within 3 mm of each other and one fish had a upper caudal fin clip, and the other a lower caudal fin clip (marking pattern was not treatment-specific).p < 0.05.The behavioral assay followed protocols described by Martinovic et al. . BrieflyThe authors declare that they have no competing interests.NGR: RNA extraction, microarrays, drafted the manuscript; IRA: conceived of the study, and participated in its design and coordination and helped draft the manuscript; DM: behavior assays, helped draft the manuscript; LL: bioinformatics analysis, helped drafted the manuscript; NDD: conceived of the study, and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.functional clustering of enriched GO biological processes altered in site # 7.Click here for filefunctional clustering of enriched GO biological processes altered in site # 12.Click here for filefunctional clustering of enriched GO biological processes altered in site # 13.Click here for filefunctional clustering of enriched GO biological processes altered in site # 21.Click here for file


Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.Our study suggests that during  Yersinia comprises Yersinia pestis, the causative agent of plague, and Yersinia enterocolitica and Yersinia pseudotuberculosis, both of which cause gastrointestinal disease. Like many other gram negative bacteria yersiniae utilize a so called type III-secretion system (TTSS) to inject proteins into host cells Yersinia outer proteins”, Yops) subvert the immune cells, which permits extracellular persistence and proliferation of Yersinia. Six different Yops, YopE, YopT, YopO/YpkA, YopH, YopP/YopJ and YopM, are translocated and attack diverse aspects of cellular immunity which disturbs immune cell function at different levels Yersiniae are gram-negative bacteria which belong to the family of the Enterobacteriaceae. The genus in vivo virulence of yersiniae. Deletion of the yopM gene in pathogenic yersiniae results in a dramatic loss of virulence Yersinia infection In contrast, the intracellular actions of YopM remained largely obscure. Nevertheless, YopM has an important role for in vivo function of the individual RSK-isoforms is not well established. Inactivating mutations in the RSK2 gene in humans cause the Coffin-Lowry syndrome First insights into the cellular actions of YopM came from a study by McDonald et al., who co-immunoprecipitated the kinases RSK1 and PKN2 with YopM from transfected cells Using physiological infection conditions, our present study confirms an earlier report that YopM forms a complex with members of the RSK and PKN kinase families. We found that YopM induces long lasting activation of RSK even in the absence of upstream ERK-signalling. In vivo and in vitro evidence is provided that YopM interferes with dephosphorylation of RSK most likely by shielding it from the action of phosphatases. Hence, we can provide a novel mode of action of a bacterial effector protein explaining how hyperphosphorylation and overactivation of RSKfamily kinases is achieved.Anti-flag was from Sigma and used in the dilution 1∶3000, anti-myc clone 9E10 was from Cell Signaling and used in the dilution 1∶1000, anti-HA high affinity was from Roche and used in the dilution 1∶1000, anti-phospho-S380RSK was from Cell signaling and used in the dilution 1∶1000, anti-phospho-S221RSK was from R&D systems and used in the dilution 1∶2000, anti-RSK1 was from Santa Cruz Bitotechnology and used in the dilution 1∶1000, anti-ERK1/2 was from Cell Signaling and used in the dilution 1∶1000, anti phospho-ERK1/2 was from Cell Signaling and used in the dilution 1∶1000, anti-YopM was a gift from Jürgen Heesemann and used in the dilution 1∶3000. Anti-actin was from Millipore and used in a dilution of 1∶1000. Secondary antibodies against rabbit-IgG, rat-IgG and mouse-IgG were all from GE Healthcare and Horse radish peroxidase conjugated. All used in a dilution of 1∶10000.Yersinia enterocolitica WA Serotype O:8 YopM gene of the virulence plasmid pYV127/90; gi: 28372983) in pACYC184 AGGTACCGCTAGCCATGGCATACCCATACGACGTCCCAGACTACATGCCGCTGGCGCAGCTGGCGGA and TCTACCAAGATATCACGTTCCA: This fragment was digested with KpnI and EcoRV and cloned into the similarly digested gi:109730041. From there the whole construct was cut out with NheI and NotI and cloned into the similarly digested vector pCI-neo. Myc-YopM was created by amplifying YopM from YopM in pACYC184 mentioned above with the primers Forward CTGACCATGGAATATGGTTTTGTTTGCAATGAA and Reverse GTCACTCGAGCTACTCAAAAACATCATCTTCAAG. Subsequently, it was cloned via the restriction sites NcoI and XhoI into the pCS2+MT vector. For bacterial expression of Glutothione-S-transferase- (GST-) tagged YopM, YopM was amplified from YopM in pACYC184 using the primers Forward AGGATCCATGTATGGTTTTGTTTGCAATGAA and Reverse GAAGCTTTCTACTCAAAAACATCATCTTCAAG and subsequently cloned into the pGEX-KG vector BamHI and HindIII. YopM-CBP-SBP in pACYC184 was created by amplification of a fragment of YopM with the primers ATTGATCCATATGAATTTGCTCAT and TTCTAGAGTACGCGGCCGCCTCAAAAACATCATCTTCAAG, digestion with NdeI/XbaI and ligation into YopM in pACYC184 which had also been digested with NdeI and XbaI. This resulted in a YopM without a stop codon and a 3′-NotI-restriction site. The tandem-affinity-tag containing the Calmodulin-Binding-Peptide (CBP) GATCCTAATTAATTAAG; oligo-R: GATCCTTAATTAATTAG) were inserted into the BamHI site of pNTAP. Afterwards, an XbaI-linker (New England Biolabs) was inserted into the EcoRV site of pNTAP. The resulting pNTAP and the YopM-pACYC184 construct without stop codon and the additional NotI site described above were then digested by NotI and XbaI and the insert from pNTAP was ligated into the YopM-pACYC184 construct resulting in a YopM C-terminally tagged with the two consecutive affinity tags CBP and SBP from the pNTAP vector.The following expression plasmids have been described previously: HA-tagged RSK1 MEK1/2-inhibitor thio)methylene)-2-(trifluoromethyl)benzeneacetonitrile) and HA1077 were all purchased from Calbiochem, rotenone, 2-deoxyglucose and Flag-sepharose were purchased from Sigma.2 in DMEM with 10% FCS containing 100 IU/ml Penicillin and 100 µg/ml Streptomycin . Transfection was carried out at approximately 50% confluence using the Calcium phosphate method following standard procedures. During serum starvation conditions HEK293T cells were incubated in DMEM as described above without FCS for 12 hours. For serum starvation medium was exchanged 16–24 hours before harvesting to serum-free DMEM with 100 IU/ml Penicillin and 100 µg/ml Streptomycin. For MEK1/2 inhibition 48 hours after transfection HEK293T cells were serum-starved and 16–24 hours later were treated with 6 µM MEK-inhibitor . 60 minutes later cells were harvested. For ATP-depletion of HEK293T cells the medium was exchanged for PBS (Gibco) containing 10 mM 2-deoxyglucose and 5 µM rotenone (both from Sigma) HEK293T (ATCC# CRL-11268) cells were grown on 100 mm or 35 mm tissue culture dishes (Nunc) at 37°C and 5% COYersinia strains and Infection – Yersinia enterocolitica strains used in this study are deltaYopM, a derivative of the Yersinia enterocolitica Serotype O:8 strain WA-314 harbouring the virulence plasmid pYVO8 plasmids) (deltaYopM(pYopM-CBP-SBP)), deltaYopM complemented with YopM in pACYC184 Yersinia enterocolitica strain WA-C Yersinia cultures were grown overnight at 27°C and then diluted 1∶20 in fresh Luria-Bertani broth and grown for another 2 h at 37°C in order to induce activation of the Yersinia type III secretion machinery and expression of Yops. Bacteria were centrifuged and resuspended in ice-cold PBS. J774A.1 cells were then infected by addition of bacteria to the antibiotic free medium at a multiplicity-of-infection of about 50∶1. 90 min after infection of cells the bacteria were killed by the addition of gentamicin (100 µg/ml). Cells were harvested and lysed in phosphatase inhibition buffer ) at the indicated time points.8 cells) were infected with deltaYopM(pYopM-CBP-SBP) as described above. 90 min after infection the cells were washed 3 times with PBS and were then harvested and lysed in 10 ml of the lysis buffer supplied with the Interplay™ Tandem Affinity Purification kit (Stratagene) by three freeze-thaw rounds. The following tandem affinity purification procedure was performed according to the instructions of the manufacturer. Briefly, the lysate was cleared by centrifugation and then incubated with streptavidin sepharose for 2 hours at 4°C under constant rotation, washed three times with the provided streptavidin binding buffer and was then eluted with streptavidin elution buffer. This eluate was then incubated with calmodulin sepharose for another 2 hours at 4°C and subsequently washed again three times with calmodulin binding buffer. The washed beads were boiled for 10 min in 2x SDS-loading buffer and loaded onto a SDS-PAGE. Afterwards, the gel was Coomassie stained and the visible bands cut out and analyzed by mass spectrometry.Ten 100 mm culture dishes of J774A.1 cells . After digestion the gel pieces were repeatedly extracted (50% acetonitrile/5% formic acid), the combined extracts dried down in a vacuum concentrator and redissolved in 5% methanol/5% formic acid. 0.5 µl of sample was mixed with an equal volume of matrix solution (saturated solution of cyano-4-hydroxycinnamic acid (HCCA) in 65% water/35% acetonitrile/0.1% trifluor acetic acid (TFA)) and applied onto a MALDI target by the dried-droplet method. Peptide mass fingerprint data were determined on a MALDI-TOF mass spectrometer  in reflector mode. Database searches were done with the Mascot search algorithm version 2.2  using the following parameter: mass tolerance: 50 ppm, one missed tryptic cleavage allowed, fixed modification: carbamidomethyl cysteine, variable modification: monooxidized methionine, database searched: NCBI nr 20100116, searches limited to mus musculus.The proteins (bands cut out from Coomassie stained SDS gels) were reduced with 10 mM dithiothreitol (DTT) at 56°C for 30 min, the cysteine residues modified with iodacetamid  and the protein in-gel digested with trypsin  in 50 mM NHd-galctoside (IPTG) was added to a final concentration of 1 mM and cultures were incubated for further four hours. Bacteria were then harvested by centrifugation, resuspended in PBS with 1x Complete protease inhibitor cocktail (Roche) and lysed by ultrasonification. After pelleting the bacterial debris by centrifugation, the supernatant was aliquoted and stored at −80°C. Bacterial lysates were then used either for purification or for pull-down assays. In order to purify GST or GST-YopM bacterial lysates were poured onto a column loaded with 0.5 ml Glutathion-sepharose resin . After binding the column was washed with 10 ml PBS +1% Triton-X100 and subsequently with 10 ml PBS. Proteins were eluted from the sepharose in 5 ml 100 mM NaCl, 50 mM Tris pH 7.5, 20 mM Glutathion (Sigma) and 1x Complete protease inhibitor cocktail (Roche). Protein amounts in the eluates were quantified by DC protein assay (Biorad). GST-pull-down assays were essentially done as described 35S]-methionine  labeled in vitro translated  proteins were added. After overnight incubation, the sepharose was washed eight times in binding buffer, boiled in 2x SDS-loading buffer and loaded onto a SDS-PAGE. Bound proteins were visualized by autoradiography.Bacterial protein expression and purification were done essentially as described Cellular lysates were prepared by three consecutive freeze/thaw cycles in phosphatase inhibition buffer ). Lysates were then resolved in 10% SDS polyacrylamide gels and afterwards, proteins were transferred to PVDF membranes by electroblotting. Membranes were blocked in 5% milk powder in PBS +0.1% Tween for 1 h and subsequently incubated with the indicated first antibodies diluted in blocking solution overnight at 4°C. After washing three times in PBS +0.1% Tween, membranes were incubated in the appropriate second antibody for 2 h at room temperature, washed three times as above and bands were visualized by the ECL system . For the quantification of Westernblots femtoLUCENT™ PLUS-HRP (G-Biosciences) was used as chemiluminescence reagent. Chemiluminescence was recorded with the LAS 4000 mini Luminescent Image Analyzer (Fujifilm Corporation) and signals were analyzed using the Multi Gauge V3.2 software.HEK293T cells were co-transfected with the indicated plasmids. Cells were lysed 48 hours after transfection in PBS +1% Triton X-100 supplemented with Complete protease inhibitor cocktail by three consecutive freeze/thaw cycles. After clearing of the lysate by centrifugation lysates were incubated with 20 µl Flag-sepharose (Sigma) overnight. Afterwards the sepharose was washed eight times with 1 ml lysis buffer and then resuspended in 2x SDS-buffer, boiled and loaded onto SDS-PAGE. After western blotting, the membrane was incubated with the indicated antibodies.50 ng active RSK, ERK2 and MEK1 (Millipore) were preincubated with 1 µg of bacterially expressed GST or GST-YopM in lambda phosphatase buffer (New England Biolabs) supplemented with Complete protease inhibitor cocktail (Roche) in a total volume of 20 µl for 15 minutes on ice. Subsequently, 2000 units (0.5 µl) lambda phosphatase (New England Biolabs) were added and the reactions were incubated for 5 min for p-S380RSK and 60 min for p-S221RSK, ERK2 and MEK1 at 30°C under constant shaking. Reactions were stopped by adding 20 µl 2x SDS buffer and boiling for 10 min. 20 µl of each reaction were then loaded onto a SDS-PAGE and analyzed by western blotting.Yersinia. For this purpose the coding region of the Yersinia enterocolitica Serotype O:8 strain WA-314 YopM gene was fused N-terminally to a Tandem-affinity-Purification tag (TAP-tag), which consists of two separate affinity tags, one calmodulin binding peptide (CBP) and one streptavidin binding peptide (SBP), which can be specifically eluted from their affinity matrices with EGTA and Biotin, respectively. The fusion-construct was transformed into a Yersinia enterocolitica O8 strain in which the YopM gene had been deleted, WA-deltaYopM, resulting in the complemented strain WA-deltaYopM(pYopM-CBP-SBP) (A previous study identified PKN2 and RSK1 as interaction partners of YopM CBP-SBP) . This coCBP-SBP) . While nCBP-SBP) . The intCBP-SBP) .35S]-methionine labeled PKN1, PKN2, RSK1 or RSK2. All labeled proteins bound to GST-YopM but not to GST alone  with lambda phosphatase in the presence of bacterially expressed and purified GST or GST-YopM. RSK1 was readily dephosphorylated by lambda phosphatase on serine 380 and serine 221 when GST was added to the reaction mix . In sharYersinia strains for these experiments. Two strains express modules constituting a functional type-three-secretion system, without (WA-C(pTTSS)) or with YopM complementation (WA-C(pTTSS+pYopM)). The other two strains were the YopM-deleted strain WA-deltaYopM and WA-deltaYopM complemented with YopM (WA-deltaYopM(pYopM)). Using backgrounds which either express other Yops (WA-deltaYopM) or not express any other Yops (WA-C(pTTSS) served to investigate whether other Yops influence the effects of YopM. Furthermore, the two different backgrounds WA-C(pTTSS) and WA-deltaYopM were used to overexpress YopM as both WA-C(pTTSS+pYopM) and WA-deltaYopM(pYopM) express YopM from a plasmid. This resulted in an about five-fold overexpression of YopM compared to wildtype strain WA-P as determined by westernblot quantification of lysates from infected cells  (Yersinia strain WA-C(pTTSS+pYopM)  backgrounds indicating that other Yops do not influence YopM effects on RSK phosphorylation. Noteworthy, under our experimental condition we did not observe significant inhibition of ERK1/2 by YopP, which is present in the WA-deltaYopM but not in the WA-C(pTTSS) background. Competition for translocation due to overexpression of YopM may be one explanation, though we still observed YopP-induced apoptosis at later time points in the J774A.1 cells indicating YopP translocation at effective levels. Also, different levels of phagocytosis resistance obviously do not result in differences between the WA-deltaYopM or WA-C(pTTSS) backgrounds, probably due to the rapidity of type three secretion mediated effector injection.In contrast to the HEK293T cells, basal serine 221 and serine 380 phosphorylation levels of RSK in J774A.1 cells were low under resting conditions . Upon inTaken together, we conclude from the experiments described above that YopM protects RSK from dephosphorylation leading to its sustained activation.Yersinia strains deficient for binding to RSK1 All intracellular Yops exert their effects on the cell by interacting with cellular proteins and modifying their activity The finding that YopM interacts with all members of the RSK and the PKN family raises the question whether YopM exploits the physiological functions of these kinases or whether these kinases are hijacked and directed towards novel functional contexts. With the exception of Rsk2, knockout mice have not been published for RSK or PKN family members, thus no definite statement can be made concerning redundancy, but some form of specificity for these kinases seems very likely. Thus, interaction of YopM with several isoforms may suggest, that no specific function of RSK may be targeted. Furthermore, up to now we did not find alterations in the phosphorylation status of diverse known endogenous targets of RSK (data not shown). PKN targets have not yet been investigated by us. Previous work suggested that PKN2 is not directly activated by YopM but indirectly by the overactivated RSK1 in vitro and in vivo. It is currently not exactly known, which phosphatases are involved in the dephosphorylation of RSK, but the rapidity of dephosphorylation when upstream ERK signalling is interrupted suggests a tightly regulated system. Only two phosphatases, PP2Cdelta Interaction of YopM with RSK1 has been shown to activate RSK1 as analyzed by kinase assays It is currently unclear how yersiniae may benefit from sustained activation of RSK during infection. As already discussed above, activation of RSK may only serve the subsequent activation of PKN as we and others were so far unsuccessful in identifying changes in the phosphorylation status of some of the endogenous substrates of RSK  or were infected with deltaYopM(pYopM-CBP-SBP) (lanes 2–4 left and right panel)and lysates were prepared 90 minutes after infection. Lysates were then incubated with 20 µl of Streptavidine-sepharose (lanes 1 and 2 left and right panel) or Protein A/G sepharose (lanes 3 and 4 left and right panel) and the precipitates and aliquots of the lysates before immunoprecipitation were analyzed by western blotting with the indicated antibodies.(0.57 MB PDF)Click here for additional data file.Figure S2Analysis of YopM quantities translocated into J774.1 cells by different Yersinia strains. J774.1 cell were infected with WA-P, WA-deltaYopM, WA-deltaYopM(pYopM), WA-C(pTTSS) and WA-C(pTTSS+YopM) and harvested after 90 min. Cellular lysates were then subjected to western blotting and probed with the indicated antibodies.(0.35 MB PDF)Click here for additional data file.


The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies. Constitutive activity of BRAF is known to be critical for the proliferation and survival of melanoma cells through the activation of the RAF/MEK/ERK mitogen activated protein (MAP) kinase pathway . In lighHuman melanoma cells were isolated and cultured as described in μM) CI-1040 or LY294002 in triplicate. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays were performed as described previously  (0.01–30 Proteins were extracted and blotted as described in Melanoma cells were prepared and stained as described previously (Comparative genomic hybridization (CGH) and mutation sequencing were performed as described previously  to 10 μM (for C8161) (50 for growth inhibition (280 nM), did not show substantial reduction in pERK phosphorylation until 3 μM U0126  . When weμM U0126 . In the μM U0126 . To explM U0126  of these melanoma lines harboured the V600E mutation in BRAF. Others have demonstrated the presence of amplifications in downstream components of the cell cycle machinery such as cyclin D1 and CDK4, which may contribute to this observed resistance . The planning of future clinical trials would likely benefit from the inclusion of both a phospho-specific signal transduction marker (such as pERK) and a proliferation marker (such as Ki67) to allow a true measure of treatment progress at the level of the tumour.


The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2.We found that in a dual (MK2 and ATF2) substrate assay, MK2-p38 interaction reduced the activity of p38 against ATF2. We further constructed a detailed kinetic mechanistic model of p38 phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called 'substrate-selective' p38 inhibitors in the dual substrate assay. Importantly, it was found that excess MK2 results in a stoichiometric effect in which the formation of p38-MK2-inhibitor complex prevents the phosphorylation of ATF2, despite the preference of the compound for the p38-MK2 complex over the p38-ATF2 complex. MK2 and p38 protein expression levels were quantified in U937, Thp-1 and PBMCs and found that [MK2] > [p38].Our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. We find that, given our current understanding, it is unlikely that 'substrate-selective' inhibitors of p38 will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of MK2 relative to p38. The precedence for tumor necrosis factor alpha (TNF) as a target has been well established by the anti-TNF biological therapeutics currently on the market . While tDozens of small molecule p38 inhibitors have been put into the clinic for the treatment of chronic inflammatory diseases such as RA -9. Theseet al. a 'substrate-selective' a p38-alpha inhibitor was described that prevented p38-alpha-dependent MK2 phosphorylation  but did not prevent phosphorylation of another p38 substrate, ATF-2   > [p38] in our assay design, all of the active p38 will be sequestered into the p38-MK2-inhibitor complex, reducing the pool of active p38 that is free to phosphorylate ATF2, in spite of the apparent 'substrate-selective' behavior seen in the single assays.The modest left-shift predicted and experimentally observed in phospho-ATF2 ICin silico and biochemically, that the presence of additional substrates results in the loss of substrate selectivity. Due to the sequestration effect of substrate selective compounds, we have found that a necessary condition for this to take place in vivo is that [active p38] < [MK2]. In order to see if this holds in relevant cell types, we measured protein expression levels of p38 and MK2 in the PMA-activated U937 and Thp-1 monocytic cell lines as well as in primary peripheral blood mononuclear cells (PBMCs). Protein expression was measured via Western blot on a per cell basis using recombinant standards and quantifying bands via densitometry, shown in Figure in situ [active p38] < [MK2]. This is also consistent with other reports of protein expression levels seen in MAPK signaling cascades [Thus far, we have demonstrated both cascades . Under tThe concept of a 'substrate-selective' inhibitor as a means to avoid unwanted side-effects is a very attractive one. The use of multiple screening assays to identify such compounds is a convenient and efficient method for identifying chemical entities with specific effects. However, great care should be taken to understand the cellular target(s) to determine the feasibility of such strategies particularly in more complex environments. From this work we specifically sought to explore the parameters governing the effectiveness of the substrate-selective inhibitor strategy in general and in particular for the p38-MK2 system.On the path to understanding the behavior of substrate-selective inhibitors, an additional mechanism was uncovered: following interaction with MK2, the activity of p38 with regard to ATF2 is substantially reduced. From our analysis, there are multiple mechanisms that could give rise to this, including alteration of the affinity for ATF2 or the catalytic rate constant. Further determination of kinetic mechanism and molecular details was beyond the scope of this work. One might hypothesize that MK2 may, in some way, be eliciting an inhibitory phosphorylation on p38, however, this remains to be demonstrated.Given that MK2 already has a much higher affinity for p38 than ATF2, one may ask how ATF2 would get phosphorylated at all within the cell . In this case, one must recall that these are competing kinetic processes, rather than static events. Our dual substrate assay time course Figure  confirmsin vivo. Additionally, this mechanism of sequestration-mediated inhibition of secondary substrates would extend to other substrates than ATF2 as well. It is worth noting that the presence of scaffolding proteins and higher-order interactions taking place in the cell that may locally alter protein concentration and drive interaction that would otherwise not take place in free solution. One cannot explicitly model such effects, however, given that their purpose is to locally increase protein concentration it is unlikely to change the outcome of our analysis.We have used our computational model to predict that the introduction of multiple substrates would result in the loss of substrate selectivity and experimentally validated this finding in a biochemical assay. Alternately stated, the addition of MK2 to the p38-ATF2 reaction was able to make CMPD1 a potent inhibitor of ATF2 phosphorylation. Through the construction of a kinetic model of the proposed mechanism of action we demonstrate that these findings are a general result and not a compound-specific finding. Our analysis demonstrated that relative p38 and MK2 levels play a defining role in determining that the substrate-selective mechanism is not likely to work as intended In our kinetic model, we modeled each phosphorylation event as a one-hit reaction, even though p38 is known to phosphorylate MK2 and ATF2 at multiple sites . This was done to restrict the number of kinetic parameters in the model to as few as needed to adequately recapture the overall system behavior and be consistent with the experimentally measurable quantities, namely single phosphorylation sites.Conceptually, one could consider p38-mediated phosphorylation or activation as the process by which p38 comes into contact with one of its substrates and performs the necessary modifications, either via one single-step reaction, or multiple dissociation and rebinding events. In order to perturb this process a compound needs to interfere with only one of these events. Our experimental data confirm that our compounds are able to interfere with MK2 and/or ATF2 phosphorylation, even though only single site was measured. If a substrate-selective inhibitor stabilizes the p38-MK2 interaction (or one of many possible forms) it will effectively sequester and prevent p38 from performing ATF2 phosphorylation, regardless of the number of phosphorylation sites. Consequently, the single phosphorylation site kinetic model was sufficient to describe and predict the observed behavior of both classical and substrate-selective model.To further address the question of how multiple phosphorylation sites may affect a substrate-selective inhibitor we constructed a simplified model incorporating varying numbers of ATF2 and MK2 phosphorylation sites .The ability of active p38 to phosphorylate ATF2, MK2 or both was tested in a biochemical assay. Reagents were added in the following order: compound, substrate(s), ATP, p38 for a final reaction volume of 40 ul in low protein binding 96-well plates (Immulon-1B). Final reaction conditions were 0.5-5 nM p38 , 50 uM, 100 nM ATF2 (Santa Cruz Biotechnology SC-4007) 10 nM MK2 (Upstate 14-349). Reactions were performed in buffer containing 20 mM HEPES pH 7.5, 10 mM MgClPhospho-ATF2 was measured with the Meso-Scale Discovery (MSD) platform using an in-house assay. High-binding MSD plates were spotted with 5 ul of 25 ug/ml rabbit anti-ATF2  and left to dry overnight. Plates were blocked with 3% MSD Blocker A in MSD Tris Wash Buffer for at least 1 hr at RT. 25 ul of sample was added with 25 ul of antibody cocktail containing 0.2 ug/ml mouse anti-phospho ATF2 (Santa Cruz Biotechnology SC-8398) and 1 ug/ml goat anti-mouse sulfo-tag (MSD R32AC-1) diluted in 1% Blocker A in Tris Wash. Plates were incubated for > 3 hr at RT, washed 4× with 150 ul MSD Tris Wash Buffer and read on the Meso-Scale Discovery Sector imager using 2× MSD Read Buffer T.Phospho-MK2 was measured with the MSD platform using an in-house assay. High-binding MSD plates were spotted with 5 ul of 25 ug/ml goat anti-MK2  and left to dry overnight. Plates were blocked with 3% MSD Blocker A in MSD Tris Wash Buffer for at least 1 hr at RT. 25 ul of sample was added with 25 ul of antibody cocktail containing 1 ug/ml mouse anti-phospho MK3  and 1 ug/ml goat anti-rabbit sulfo-tag (MSD R32AB-1) diluted in 1% Blocker A in Tris Wash Buffer. Plates were incubated for > 3 hr at RT, washed 4× with 150 ul MSD Tris Wash Buffer and read on the Meso-Scale Discovery Sector imager using 2× MSD Read Buffer T.Anti-total p38  at 1 ug/ml was bound to Immobilized Protein G (Pierce 20398). 5 nM p38 was incubated with varying amount of MK2 for 1 hour at RT and then added to p38 antibody bound to protein G and incubated o/n at 4°C. The next day antibody and protein G were washed once with enzyme buffer, once with IP Buffer  and twice more with enzyme buffer ending in fresh enzyme buffer for the second kinase reaction with ATF2. After 1 hour stop buffer was added and assayed for phospho-ATF2 in the MSD assay.U937 and Thp-1 cells were obtained from ATCC and cultured under the recommended conditions. Prior to cell plating, cells were differentiated into macrophages with PMA (Sigma). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll gradient centrifugation and maintained in RMPI + 10% FBS.10 and 1.34 × 1011 molecules per well for p38-alpha and MK2, respectively. p38-alpha blots were probed with rabbit anti-p38alpha . MK2 blots were probed with rabbit anti-MK2 . Blots were imaged and bands quantified using densitometry. Protein standards were used to construct a standard curve, from which the number of molecules per cell could be calculated. Numbers were converted into uM, based on reported cell volumes for the different cell types: 1.09 pl, 0.97 pl and 0.38 pl for U937, Thp-1 and PBMC, respectively [U937, Thp-1 cells and PBMCs were counted and lysed. Following total protein determination via BCA assay (Pierce), cells/ug was calculated. Cell lysates were run on SDS-PAGE gels (Invitrogen) at 2 and 5 ug total protein per well alongside a 5 point standard of recombinant protein , MK2 ). Three-fold dilutions of standards were loaded, starting at 1.68 × 10ectively -28.KD, ATP, independent of further complex formation. p38 (ATP bound or not) may reversibly complex with either ATF2 or MK2 yielding p38-ATF2, p38:ATP:ATF2, or p38:MK2, p38:ATP:MK2. Complex formation is characterized by affinities KD, ATF2 and KD, MK2. p38:ATP:ATF2 and p38:ATP:MK2 undergo a irreversible catalysis step yielding phospho-ATF2 and ADP with rate constant kcat, ATF2 or phospho-MK2 and ADP with rate constant kcat, MK2. It is known that p38 phosphorylates ATF2 on Thr69 and Thr71 in a two-step distributive mechanism [6 M-1s-1; ATP and compound forward interaction rate constants were set to 107 M-1s-1). Simulations were performed in a 40 ul reaction volume, with 0.5 nM p38, 100 nM ATF2, 10 nM MK2 and 50 uM ATP.A mass-action kinetic model was built to precisely simulate the experimental biochemical reaction conditions. In the kinetic model, activated p38 reversibly binds ATP with affinity echanism ,29 and Mechanism , howeverD, ATP is lowered 10-fold; [D, ATF2 is lowered 10-fold; and [cat, ATF2 is lowered 10-fold. Substrat10-fold;  KD, ATF2old; and  kcat, AThttp://www.sbml.org. The SBML code was translated into the Jacobian language  using a translator written in MATLAB  using the SBML toolbox http://sbml.org/software/sbmltoolbox/. Scripts were written in Jacobian and MATLAB to generate dose-response curves and figures. Model files are available in Additional Files 50's were calculated using GraphPad Prism .The kinetic model was coded in Teranode Design Suite . The completed model was exported in SBML format 50: the concentration of an inhibitor that is required for 50% inhibition of an enzyme in vitro; MSD: Meso-Scale Discovery.RA: rheumatoid arthritis; LPS: lipopolysaccharide; TNF: tumor necrosis factor alpha; ATF2: activating transcription factor 2; MK2: mitogen-activated protein kinase-activated protein kinase 2; p38: p38 MAP kinase; ICBSH is currently employed by Merrimack Pharmaceuticals. KMS is currently employed by Novartis Pharmaceuticals. JRC is currently employed by Pfizer, Inc. All authors hold stock in Pfizer, Inc.All authors hold stock in Pfizer, Inc.All authors contributed to the design of experiments. BSH and JRC constructed and analyzed the kinetic models. BSH and KMS performed the experiments. BSH drafted the manuscript. All authors read and approved the final manuscript.Supplementary Figure S1Click here for fileSupplementary Figure S2Click here for fileSSImodel. Substrate-selective inhibitor model in SBML (systems biology markup language http://www.sbml.org) format. All model variations described in the text are included.Click here for fileSSImodel. Substrate-selective inhibitor model translated into format for Jacobian modeling software . This file includes scripts used to generate simulated data for dose-response curves.Click here for fileSSImodel_expanded. Supplementary model - expanded substrate-selective inhibitor model including multiple phosphorylation sites (used for supplementary figures). This model is in MATLAB SimBiology (R2009a) format.Click here for fileSSImodel_expanded. Supplementary model, exported into SBML (systems biology markup language http://www.sbml.org) format.Click here for file


Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases  and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.Mutations in the gene encoding  Clinically, PD is characterized by a movement disorder that includes resting-tremor, muscular rigidity, and bradykinesia, as well as non-motor symptoms. Neuropathologically, selective loss of dopaminergic (DA) neurons in the LRRK2 (Leucine-rich repeat kinase 2) are the most common cause of inherited PD LRRK2-related PD are pathologically similar to sporadic disease. LRRK2 encodes for a large, 286 kDa, multidomain protein Dominant mutations in the gene +2-binding motif (DYG) of the kinase domain, and has been shown to increase the kinase activity of LRKK2 in vitro, it is unclear whether autophosphorylation is physiologically relevant. An alternative to autophosphorylation as an assay for kinase activity is to use generic substrates such as myelin basic protein (MBP) The most prevalent LRRK2 mutation, G2019S Several heterologous LRRK2 substrates have been suggested for LRRK2 including moesin in vivo using Drosophila models. Similarly, Tain et al have shown that phospho-4E-BP levels are decreased in a homozygous knockout model of drosophila LRRK Drosophila homologue, dLRRK. However, the kinetics of the phosphorylation reaction have not yet been reported. In the present study we investigated the kinetic properties of 4E-BP phosphorylation by LRRK2 compared to autophosphorylation activity and we used a LRRK2 inducible cellular model as well as transient expression in HEK cells to measure the effect of LRRK2 expression on 4E-BP activation at T37/T46.Recently Imai et al. reported 4E-BP as a potential substrate of LRRK2 in vitro  were incubated with 12 nM of LRRK2, and the incorporation of 33P over the time-course of reaction was measured .After confirming that 4E-BP is an measured . The effmeasured . Howeverversus 1.5 to 12 µM 4E-BP), which is apparent on examination of a single exposure with both reactions  compared to LRRK2-wild type . However, Km for 4E-BP was similar for both wild type LRRK2 and MAPK14/p38α  (We next considered whether kinases other than LRRK2 could mediate the same reaction picking three kinases from different families in the kinome . Under cctively) .33P was measured by scintillation counting. We observed that 1 mole of ATP was incorporated into 70 moles of LRRK2 whereas 1 mole of ATP was incorporated into 1.58×103 moles of 4E-BP after 60 minutes of reaction suggesting that LRRK2 autophosphorylation is approximately 20 times more efficient than 4E-BP phosphorylation under the same conditions. To determine the stoichiometry of LRRK2 autophosphorylation compared to 4E-BP phosphorylation, 12 µM of 4E-BP was incubated with 12 nM of LRRK2, and the incorporation of Next we assessed the effect of expression of LRRK2 in inducible stable HEK 293FT clones. Although we were able to readily detect the phospho-4E-BP T37/T46, S65 and T70 epitope in these cells , we did Finally we transiently overexpressed LRRK2-wild type, LRKK2-G2019S, LRRK2-R1441C and MAPK14/P38α in HEK 293FT cells. We did not detect any alteration in 4E-BP phosphorylation either by wild type or mutant LRRK2, although we found that MAPK14/P38α could phosphorylate 4E-BP under the same conditions .LRRK2 gene represent the most common cause of PD LRRK2-linked molecular pathogenesis. Therefore, in the present study we have investigated the kinase activity of LRRK2 with respect to its autophosphorylation as well as phosphorylation of 4E-BP, a novel reported LRRK2 substrate. We were able to replicate some previous experiments showing that 4E-BP can be phosphorylated by LRRK2 in vitro but we found that this activity is weak compared to autophosphorylation. Moreover, using inducible LRRK2 cells or transient transfections, we could not detect any appreciable increased in 4E-BP phosphorylation after LRRK2 expression. However, we observed that MAPK14/p38α increases 4E-BP phosphorylation in HEK 293FT cells after transient expression, suggesting that MAPK14/P38α is a robust kinase for 4E-BP.Although mutations in the in vitro and that I2020T, a pathogenic mutation found in the original PARK8-linked family in vitro and that 4E-BP phosphorylation is enhanced when incubated with LRKK2-G2019S, in line with multiple studies consistently showing an enhanced kinase activity for this mutation. Our kinetic analysis with 4E-BP also indicates that the G2019S mutation stimulates LRRK2 activity by increasing the catalytic Vmax constant rather than enhancing substrate-kinase binding Km affinity. This supports prior suggestions that G2019S alters processivity for LRRK2 rather than substrate binding and would be consistent with the relative ease by which enhanced activity of G2019S has been demonstrated. The G2019S mutation occurs within a highly conserved activation segment of the protein and is postulated to result in a gain of function of its kinase activity, compatible with its autosomal dominant mode of transmission Prior experiments suggested that LRRK2 can phosphorylate 4E-BP 33P from labeled ATP into LRRK2 itself rather than 4E-BP, even when the later is present at more than a hundred-fold molar excess, demonstrates that the LRRK2 autophosphorylation reaction is much more efficient under these conditions. One limitation of this experiment is that a truncated LRRK2 version was used, as this construct is more soluble and active than full-length protein. However, in cell lines expressing full length LRRK2 that was active when isolated and tested in vitro for autokinase activity, we did not see any evidence of 4E-BP phosphorylation either (see below). These results were also strengthened by measuring the stoichiometry of phosphorylation for LRRK2 and 4E-BP in the same reaction, where we found that LRRK2 incorporated phosphate more efficiently than 4E-BP again despite the latter being present at molar excess.However, we also found that while LRRK2 is able to phosphorylate 4E-BP, autophosphorylation is more efficient under the conditions in which we performed these assays. This was perhaps not immediately appreciated from prior studies where the relative activities of LRRK2 autophosphorylation and LRRK2 mediated phosphorylation of 4E-BP were presented by showing each separately and in experiments where the concentration of LRRK2 is not known. However, we can facilitate a comparison between autophosphorylation and 4E-BP phosphorylation by using recombinant LRRK2 fragment of known concentration and by analyzing kinase reaction products on the same gel. Three-fold higher incorporation of in vitro. As we have not performed an analysis of the complete kinome, we cannot be certain how many kinases can phosphorylate T37/T46 of 4E-BP but because other kinases can trigger phosphorylation of this site, the measurement of it in cell or tissue lysates is not a simple measure of LRRK2 activity per se. Second, in HEK 293FT inducible cell lines and in transiently transfected HEK 293FT cells we did not find any significant 4E-BP phosphorylation changes by wild-type LRRK2 or two different pathogenic mutants G2019S and R1441C, while MAPK14/P38α was able to efficiently phosphorylate 4E-BP in these cells.Two further results give pause to the conclusion that 4E-BP is an authentic substrate for LRRK2. First, MAPK14/p38α and STK3 are capable of phosphorylating 4E-BP ex vivo. Also, if 4E-BP is a substrate for several protein kinases, like MAPK14/P38α, the contribution of LRRK2 phosphorylation may not be sufficient to discriminate 4E-BP basal phosphorylation or it may require a specific stimulus. Furthermore, we determined kinetic parameters for MAPK14/p38α mediated phosphorylation of 4E-BP compared to LRRK2, and found that MAPK14/p38α is more efficient, based on its twenty times higher Vmax value  was purchased from Perkin Elmer .Truncated recombinant LRRK2 wild-type and variants (LRRK2-G2019S and LRRK2-D1994A kinase dead) (aa 970–2527) were purchased from Invitrogen . Recombinant eIF4E protein was purchased from Origene . Monoclonal anti-GST LRRK2 antibody and monoclonal anti-V5 LRRK2 antibody were purchased from Amersham  and Invitrogen  respectively, and used at 1∶5000 for chemiluminescent immunoblotting. Antibodies against phospho 4E-BP  and non-phospho 4E-BP and eIF4E were purchased from Cell Signaling Technology .  containing ∼12 nM of truncated recombinant LRRK2 wild-type, LRRK2-G2019S or LRKK2 kinase dead with different concentrations of 4E-BP  and ATP (100 µM). Reactions were stopped by the addition of SDS sample buffer and western blots were performed after electrophoresis of samples on 4–20% Tris-HCl gels. Blots were incubated with anti GST, phospho 4E-BP  or total 4E-BP antibodies.Non-radioactive 33P]ATP (5 µCi/reaction) in kinase reaction buffer consisting of 20 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 1mM Na3VO4, 5 mM β-glycerophosphate, 0.02% Triton X-100, and 2 mM fresh dithiothreitol at 30°C in a final volume of 20 µL at five different time intervals  with four different concentrations of 4E-BP . Reactions were terminated by the addition of SDS sample buffer (Invitrogen). LRRK2 autophosphorylation and 4E-BP phosphorylation were detected by autoradiography using a phosphor screen, scanned on a STORM 840 scanner , and quantitated using IMAGE J software (NIH). The kinetic parameter Km and Vmax were determined by fitting the rate of reaction versus substrate concentration curves to the Michaelis-Menten equation, ν/Vmax = [S]/([S]+Km) using a nonlinear regression analysis in Graphpad Prism software version 4.0 . All fitted curves had R>0.9.For kinetic studies and LRRK2 autophosphorylation experiments, truncated recombinant LRRK2 wild-type, LRRK2 kinase dead and LRRK2-G2019S were incubated separately with ATP (5 µCi/reaction) in kinase reaction buffer at 30°C in a final volume of 20 µL at five different intervals of time  with 12 µM 4E-BP.We also performed similar reactions with three other kinases. STK3 (serine/threonine kinase 3), MAPK14/p38α (mitogen-activated protein kinase 14) and DAPK2 (death associated protein kinase 2) were incubated separately with [γIn vitro phosphorylation assays performed as previously described, were run on SDS-PAGE and transferred onto PVDF membranes. After ponceau staining, bands corresponding to LRRK2 or 4E-BP were excised according to their molecular weight and incorporated radioactivity was assessed by LS 6500 mulitipurpose scinitillation counter . A calibration curve was built with known amounts of radiolabeled 33P-ATP  and the number of moles of Pi (inorganic phosphates) incorporated per mole of protein was calculated by linear interpolation of the cpm (count per minute) data collected from the phosphorylated proteins.5′-ATGTCTCAGGAGAGCCCACGTTCTAC-3′ and 5′-TCAGGACTCCATCCTTCTTGGTCAAG-3′. MAPK14/P38α cDNA was subsequently cloned by gateway recombination from the donor vector pCR-GW8 (Invitrogen) into pCMV-2×Myc destination vector described by Greggio et al (29).Full-length LRRK2, LRRK2-G2019S and LRRK2-R1441C were cloned to generate N-terminal 2×-myc as described previously 2. Cells were induced by 1 µg/µl doxycycline after one day in culture. Cells were harvested 24hrs after induction. 4E-BP phosphorylation was determined by western blot after electrophoresis of samples on 4–20% Tris-HCl gels. Blots were incubated with anti V5, phospho 4E-BP  or total 4E-BP antibodies.LRRK2 wild-type, LRRK2-R1441C and LRRK2-G2019S HEK 293FT inducible cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  and penicillin (100 units/mL) and streptomycin (100 units/mL) at 37°C and 5% COFor transient transfection HEK 293FT cells were cultured as above and transfected using 40 ug of PEI (polyethyeleneimine)  for 20 ug of plasmid DNA. Cells were harvested 48hrs after transfection. 4E-BP phosphorylation was determined by western blot after electrophoresis of samples on 4–20% Tris-HCl gels. Blots were incubated with anti myc, phospho 4E-BP  or total 4E-BP antibodies.