In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na L), cell cycle regulation , and tumor angiogenesis  Signal transducer and activator of transcription 3 (Stat3) is one of a family of cytoplasmic proteins that participate as a transcription factor in the normal cellular responses to cytokines and growth factors Recent studies of Stat3 have suggested that it has critical roles in skin carcinogenesis induced by chemicals and UVB In addition to its recently discovered roles in the development and progression of skin tumors induced by two-stage carcinogenesis, Stat3 also plays an important role in UVB-mediated skin carcinogenesis In the current study, we show a critical role for three protein tyrosine phosphatases (PTPs); T-cell PTP (TC-PTP) and the Src homology 2 domain-containing PTPs, SHP1 and SHP2, in the dephosphorylation of Stat3 following UVB irradiation in skin. In addition, we show that all three PTPs are required for regulating the phosphorylation of Stat3 in skin keratinocytes in the absence of treatment with UVB. Finally, we show that nuclear translocation of TC-PTP occurs following UVB irradiation and contributes to rapid dephosphorylation of activated Stat3 by this PTP. Collectively, these results suggest that Stat3 dephosphorylation by TC-PTP, SHP1, and SHP2 in skin keratinocytes following exposure to UVB irradiation plays a role as an initial protective mechanism against UVB-induced skin carcinogenesis.All experiments with animal were carried out with strict adherence to both institutional and National Institutes of Health (NIH) guidelines for minimizing distress in experimental animals. All experiments involving mice were approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC).fl/fl) has previously been described Development of TC-PTP-deficient (TC-PTP-/-) and skin specific Stat3-deficient mice  2 in keratinocyte growth medium  containing 1% fetal bovine serum until 80 to 85% confluent at which time cells were irradiated with UVB at 800 J/m2. Prior to UVB irradiation, cultured keratinocytes were washed with DPBS two times to remove all remaining medium. A small volume of DPBS was added to cover keratinocytes under a thin layer of DPBS. Under this condition, cells were irradiated with UVB. For control keratinocytes, keratinocytes were treated identically, except for UVB irradiation. After UVB irradiation, DPBS was immediately removed and prewarmed medium was added to keratinocytes for additional culture before harvest.A previously described method Skin epidermis, primary keratinocytes, and 3PC keratinocytes (control and UVB treated) were used to prepare total cell lysates. For epidermal protein, mouse skin samples were placed on an ice-cold glass plate, and the epidermis was removed with a razor blade and placed into RIPA buffer containing 1% Triton X-100, protease inhibitor cocktail , and phosphatase inhibitor cocktail I, II (Sigma-Aldrich Co.). For keratinocyte protein, cultured keratinocytes were washed with DPBS three times and placed into RIPA buffer by scraping. The lysates were incubated on ice for 20 min and then centrifuged at 14,000 g for 20 min at 4°C. The supernatant obtained from the high speed spin was used for the cytosolic fraction. Cytoplasmic and nuclear fractions were prepared from keratinocytes using NE-PER nuclear and cytoplasmic extraction reagents .L, anti-SHP1, anti-SHP2 , anti-phospho-Stat5A/B , anti-Stat5 , anti-TCPTP 3E2 Twenty-five to fifty µg of total protein from mouse epidermis or keratinocytes (control and UVB treated) were resolved using SDS-PAGE. For cytoplasmic and nuclear fractions, 30 µg of cytoplasmic protein or 10 µg of nuclear protein were resolved using SDS-PAGE. The samples were transferred onto a nitrocellulose membrane using an electroblotting method. The membrane was incubated overnight at 4°C with primary antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Chemiluminescent detection reagents  were used to detect immunoreactive protein. The following primary antibodies were used: anti-phospho-Stat3 (Tyr705), anti-Stat3, anti-phospho-Stat1 (Tyr701), anti-Stat1, anti-cyclin D1, anti-Bcl-x2. One or two hours after UVB irradiation, keratinocytes were fixed with 100% methanol at −20°C for 15 min. After permeabilization with 0.5% Triton X-100 and blocking, cells were incubated with a monoclonal anti-TC-PTP antibody (R&D Systems Inc.) at 4°C for 12 h. Following extensive washing with PBS, cells were then incubated with Cy3-conjugated anti-mouse secondary antibody  at 37°C for 1 h and mounted onto glass slides in Vectashield mounting medium with DAPI. For transfection, TC45-WT and TC45-D182A/Q262N substrate-trapping mutant were generated as described before 2. Two hours after UVB irradiation, keratinocytes were fixed with 100% methanol at -20°C for 15 min. Exogenous TC45 expression was detected by anti-myc antibody .Primary keratinocytes were grown on plastic chamber slides and were irradiated with UVB at 800 J/m2.3PC keratinocytes were grown overnight to ∼40% confluence and transfected with ON-TARGETplus SMART pool short interfering RNA (siRNA) specific for mouse TC-PTP, , PTPRT, SHP1, SHP2, or ON-TARGETplus siCONTROL Non-targeting pool siRNA as a control . Transfection was performed with Lipofectamine RNAiMAX (Invitrogen). Forty eight hours of siRNA transfection, cells were irradiated with UVB at 800 J/m5′-AGTTCCAGCCTGCCATCCG-3′ and 5′-TCCAAAAGTCCTTCACCGTCT-3′ which are located in exons 16 and 20. The expected length of PCR product was 327 bp. β-casein was used as a control.Total RNA was isolated from 3PC keratinocytes (control and UVB treated) according to the manufacturer's instructions and then further purified by Rneasy columns . RNA was resuspended in diethyl pyrocarbonate-treated water and quantified by spectrophotometry. One µg of RNA was reverse transcribed to cDNA by random priming. PTPRT expression was examined by PCR with primers 3VO4 for 12 h before UVB exposure significantly increased Stat3 phosphorylation at 3 h (180 min) compared to the level found in untreated keratinocytes  involved in Stat3 dephosphorylation in skin keratinocytes in response to UVB irradiation, knockdown of each of the above mentioned PTPs was performed using RNAi. As shown in Stat3 localizes in the cytoplasm as an inactive form. After activation, tyrosine phosphorylated Stat3 translocates to the nucleus. Therefore, the cellular localization of TC45, SHP1 and SHP2 was investigated in keratinocytes following UVB irradiation. As expected, both SHP1 and SHP2 were predominantly localized in the cytoplasm compared to nucleus . Their cIt is possible that growth factors (e.g. TGFα) might increase nuclear translocation of TC45 because Stat3 is activated by growth factor signaling in keratinocytes Transfection analysis using both TC45-WT and the substrate-trapping mutant TC45-D182A/Q262N, lacking phosphatase activity by substituting its essential catalytic residues, confirmed its nuclear translocation following UVB exposure. In 3PC keratinocytes expressing TC45-WT, TC45 is primarily localized to cytoplasm and translocated to the nucleus following UVB exposure . The TC4Studies using keratinocytes from TC-PTP-deficient mice TC45, SHP1 and SHP2, are involved in UVB-mediated Stat3 dephosphorylation, respectively  were irradiated with UVB. The levels of cyclin D1, c-Myc, Bcl-xL proteins were decreased in the epidermis of untreated K5Cre.Stat3fl/fl mice compared to control wild-type mice as previously shown (fl/fl mice after UVB irradiation (L) were not increased at later time points following UVB exposure  on the phosphorylation changes observed in Stat3.Following irradiation of mouse skin with UVB, the level of phosphorylated Stat3 was initially decreased and then increased significantly at later time points in epidermis . Similarnd c-Myc . These lly shown  [10], [1adiation , althougexposure , indicatpidermis . CollectTyrosine phosphorylation of proteins catalyzed by protein tyrosine kinases (PTKs) plays a critical role in regulating proliferation, differentiation, and survival, and abnormalities in protein phosphorylation are associated with various human diseases including cancer. Tyrosine phosphorylation by PTKs is balanced by PTPs that regulate the rate and duration of the signaling responses that are amplified by PTKs In the current study, we show that Stat3 is rapidly dephosphorylated in both nucleus and cytoplasm of keratinocytes upon UVB exposure. TC-PTP, SHP1, and SHP2 are the primary PTPs that contribute to rapid Stat3 dephosphorylation in response to UVB irradiation in skin keratinocytes. We show for the first time that TC45 is mainly localized to cytoplasm of both mouse and human keratinocytes. In addition, nuclear translocation of TC45 contributed to UVB-mediated dephosphorylation of Stat3 in mouse keratinocytes. Finally, all three PTPs also appear to play a role in normal regulation of Stat3 phosphorylation in unexposed keratinocytes. In our previous studies, we showed that the level and/or activation state of Stat3 dramatically influenced the response of keratinocytes to UVB. In this regard, Stat3 deficiency enhanced UVB-induced apoptosis whereas Stat3 activation  reduced UVB-induced apoptosis in mouse keratinocytes in culture or mouse keratinocytes in vivo As shown in this study, TC45, SHP1, and SHP2 contributed to dephosphorylation of Stat3 in the absence or presence of UVB exposure. However, the results shown in Subcellular localization of PTPs is one of the critical factors in determining substrate specificity and subsequent signaling specificity, in addition to side-chain interactions between the substrates and areas flanking the PTP active site Stat3 has critical roles in cancer cell proliferation and survival and its constitutive activation is associated with a number of human tumors. However, the fact that naturally occurring mutations of Stat3 leading to its constitutive activation have not been identified implies aberrant growth factor signaling may play an important role in the constitutive activation of Stat3, rather than alterations in the gene per se Figure S1Quantification of phosphorylated Stat3 in keratinocytes following siRNA-mediated inhibition of PTP expression. Relative levels of phosphorylated Stat3 were quantified by densitometry. Results are the mean + standard deviation from three independent experiments. (A) Inhibition of TC-PTP expression by siRNA. (B) Inhibition of SHP1 expression by siRNA. (C) Inhibition of SHP2 expression by siRNA. Differences between no siRNA (or control siRNA) and each PTP siRNA were significant (p<0.05) by student t test.(0.85 MB TIF)Click here for additional data file.


Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of  In the complex process of steroidogenesis, the mitochondria is the site where the rate limiting step -cholesterol transport across the mitochondrial membranes- occurs Cholesterol transport requires specific interactions at the mitochondria between several proteins including the voltage-dependent anion channel (VDAC) 56-57 and Ser194-195, respectively The transcription of the StAR gene increases in a cAMP-dependent protein kinase (PKA)-dependent manner In summary, we have demonstrated that mitochondrial ERK1/2 is a regulator of cholesterol transport, emphasizing that the subcellular localization of MAPKs is an important mechanism in the regulation of specific cell functions.et al. and Manna and Stocco et al. which show that ERK signaling cascade inhibited gonadotropin-stimulated steroidogenesis The effects we observed with U0126 and PD98095 were not mediated by inhibition of PKA, since this enzyme remained fully active in the presence of both inhibitors (data not shown). Again in line with previously published results Active ERK1/2 was confirmed to be necessary for steroidogenesis. The overexpression of a wild-type form of ERK2 in MA-10 cells produced an increase in steroid production stimulated by submaximal concentrations of 8Br-cAMP . OverexpNext, we exposed MA-10 cells to differential stimulation with epidermal growth factor (EGF) or with cAMP. Physiologically, whereas cAMP analogues such as 8Br-cAMP maximally stimulate steroid production in Leydig cells, EGF only produces 10–20% of the maximal steroid production compared to cAMP As shown previously The immunoblots showed that after stimulation with 8Br-cAMP, phospho-ERK1/2 (pERK1/2) was located in the cytosol and mitochondria and, to a much lesser extent, in the nuclear fraction  constitutively present in the cytosol and mitochondria of MA-10 cells . InteresThe increase of mitochondrial pERK1/2 and pMEK1/2 due to cAMP action was abolished by treatment of the cells with the PKA inhibitor H89  and by PThe efficacy of the silence interference RNA (siRNA) treatment on the expression of the α isoform of the PKA catalytic subunit is demonstrated by the reduction in the cellular content of this isoform detected by western blot . As expein vivo experiments shown in in vitro  was found between amino acids 235 and 244. This site is conserved among ERK1/2 upstream kinases, MAPK phosphatases and ERKs substrates Initially, we examined StAR structure to identify consensus sequences that would allow docking to ERK1/2. A typical docking site known as the D domain (in vitro phosphorylation assay using the mature pure recombinant StAR protein (30 kDa) and two forms of ERK1 in the presence of [γ-32P]ATP. StAR was indeed phosphorylated by wild-type ERK1, but this phenomenon was not observed in the presence of the inactive mutant K71A of the kinase , Ser232 has a 90% probability of being phosphorylated, whereas the probability for Ser277 is only 5%. Moreover, Ser277 is relatively less conserved than Ser232 among species (data not shown). Ser232 (PLAGS232PS) is adjacent to the docking D domain (−2). To demonstrate that Ser232 is the phosphorylation site of StAR by ERK1/2, we analyzed both the proteolytic products and the phosphorylation of a mutated form of StAR protein.Next, we searched for the ERK1/2 phosphorylation site in StAR. Only two Ser-Pro motifs, targets for ERK1/2 phosphorylation, are detectable in the mature form of the murine StAR protein at Ser232 in the StAR protein sequence. We performed the in vitro phosphorylation assay described in 232 is the only residue that could appear phosphorylated in this fragment, the confirmation that Ser232 is the target of ERK1/2 phosphorylation came from the experiments using S232A, a mutated form of StAR, in which Ser232 was changed to Ala, a non-phosphorylable amino acid. This mutated form of StAR was used in the in vitro phosphorylation assay using active ERK1 as kinase. The mutation impeded the phosphorylation of StAR by active ERK1, confirming that this residue is indeed the target of the kinase  (To study the role of Ser (S232A) . Instead (S232A) . This is232; and d) double phosphorylation by PKA and ERK1/2 may favor StAR function.In this study we have demonstrated that a) steroidogenesis in MA-10 Leydig cells depends on the specific temporal pattern of ERK1/2 activation in mitochondria; b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in the organelles; c) active ERK1/2 interacts with StAR and leads to its phosphorylation at SerWe showed that PKA activates the constitutive mitochondrial MEK1/2 pool, a finding that accords with previous reports 316 and Asp319 of the ERK2 CD domain were observed to interact with Arg20 and Arg21 of the representative MKP3 peptide , an interaction putatively attributed in the present case to Lys235 and Lys237 of StAR (K235TK237LTWLLSI). Based on a computerized model of the ERK2-StAR complex  were described  complex , we foun232. This residue integrates the classic SP motif for phosphorylation of substrates by ERK1/2 We also confirmed that ERK1/2-StAR binding conducts to StAR phosphorylation at Ser235 in the StAR sequence and Asp319 in the ERK sequence constitute the closest interaction, separated by a short distance, approximately 11 Å , indicate that being the 37 or the 30 kDa forms the substrate for ERK1/2, the phosphorylation of StAR at Ser232 is necessary for its steroidogenic activity.Regarding the form of StAR protein that was subjected to 232-StAR phosphorylation in the assembly of this multiprotein complex and in its function. The additional negative charges in the StAR (30 kDa) molecule due to phosphorylation at Ser232 may contribute to the retention of the mature form of 30 kDa StAR in the outer mitochondrial membrane and to the formation of the multiprotein complex.The presence of a multi-protein complex in the outer mitochondrial membrane has functional repercussions for steroidogenesis. In this complex, phosphorylation of StAR by ERK1/2 is a key process for cholesterol transport. It is known that StAR works together with PBR which in turn interacts with VDAC. However, little is known about how StAR interacts with these proteins. In previous unpublished observations we detected that StAR could interact with VDAC. It will be interesting to further analyze the role of Serin vitro in accordance with the manufacturer's instructions. Constitutively active His-tagged ERK1 and PD98059 were purchased from Calbiochem . The PKA catalytic subunit was obtained from New England Biolabs . Site-directed mutagenesis on StAR wild-type cDNA was performed on a pRc/CMVi construction by GenScript . The siRNA against the PKA α catalytic subunit was obtained from Santa Cruz Biotechnology . The 3×flag-CMV7-ERK2 plasmid containing the wild-type or a mutated (H230R) variant of ERK2 was kindly provided by Dr. Melanie H. Cobb . All others reagents were commercial products of the highest grade available.Purified hCG was a kindly provided by Dr. Parlow . Anti-phospho-ERK1/2, anti-phospho-MEK1/2, anti-total MEK1/2 antibodies and U0126 were obtained from Cell Signaling ; anti-total ERK1/2 and anti-β-tubulin were purchased from Upstate Biotechnology . Anti- 39 kDa subunit of the NADH-cytochorme c reductase was obtained from Molecular Probes, Inc. . Anti-StAR was generously provided by Dr. Douglas Stocco . The antibody that recognizes the PKA α catalytic subunit  was a generous gift from Dr. Tellez Iñón . The antibody against Acot2 was generated at our laboratory The MA-10 cell line is a clonal strain of mouse Leydig tumor cells that produces progesterone (P4) rather than testosterone as the major steroid Cell transfections were performed by electroporation or using a cationic lipid reagent. MA-10 cells were transiently transfected by electroporation in accordance with already published procedures et al.E. coli. Then, several clones were screened and sense and antisense plasmids were obtained by midipreparations with the Wizard® system ; afterwards used for transfections of MA-10 cells.Full length StAR cDNA (Gen Bank accession n° BC082283) was obtained by PCR from MA-10 cells as described in Maloberti E. coli, the recombinant protein GST-StAR was purified by glutathione affinity chromatography  and subjected to thrombin proteolytic cleavage. Then, in vitro phosphorylation experiments were performed with 30 kDa StAR. The same protocol was followed to obtain a mutated form of the StAR protein (S232A) using the site-directed mutated StAR cDNA.Recombinant 30 kDA StAR protein was obtained as described before g for 10 minutes. MA-10 cells were resuspended in MSHE buffer  and subjected to mechanical disruption with 60 strokes with an insulin syringe. Then, nuclear fraction was obtained by centrifugation at 5000×g for 10 minutes. The supernatant is centrifuged at 15000×g for 30 minutes. Then, the pellet corresponding to the mitochondrial fraction was washed once and resuspended in MSHE buffer and supernatant corresponds to cytosolic fraction. Fractions were subjected to enzymatic analysis to assess their purity  were washed three times with the same buffer, boiled for 5 minutes, subjected to SDS-PAGE and immunoblot analysis.To study interaction between StAR protein and pERK, pull-down assays were carried out as indicated in Hirakawa and Ascoli 32P]ATP for 30 minutes at 30°C as described previously Phosphorylation assays of wild-type or S232A StAR were performed with wild-type His-tagged ERK1 or the catalytically inactive form of ERK1-GST (K71A) and PKA catalytic subunit. The assays were performed with 10 µCi of [γ-Proteins were separated by SDS-PAGE and electrotransferred to poly-(vinylidine difluoride) (PDVF) membranes as previously described MA-10 cells were grown to approximately 80% confluence on poly-L-lysine glass coverslips. After treatments, cells were incubated for 45 minutes at 36.5°C with 300 nM Mitotracker Red® , a specific mitochondrial dye. Then, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with blocking solution (0.3% Triton X-100 and 1% BSA in PBS) for 60 minutes at room temperature. The detailed procedure was described previously Data were analyzed by ANOVA followed by the Dunnet test.


T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds.We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays.From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells.In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells. Cellular homeostasis is maintained by the coordinated actions of kinases and phosphatases. Aberrant activation of several kinases due to overexpression, amplification or activating mutations are the underlying causes of many human pathologies like inflammation and cancer . ConversTCPTP is a non-receptor protein tyrosine phosphatase (PTP) that is expressed in all tissues throughout development . There aRecent data has suggested that the production of reactive oxygen species (ROS) is permissive for signalling by RTKs in response to stimuli . Apart fCompared to kinases, much less progress has been made in the development of new therapeutics targeting PTPs. Since PTP-1B has an important role in regulating insulin signalling, PTP-1B inhibitors targeting the active site are being developed for treatment of diabetes and obesity ,22. SincHere we have performed a high-throughput screen (HTS) with recombinant TCPTP (TC45) to identify novel activators of the enzyme. We demonstrate that six structurally distinct compounds are capable of activating TC45 in vitro and that one of these, spermidine, functions in cells to inhibit proliferation, PDGFRβ phosphorylation and VEGF-induced angiogenic sprouting in a TC45-dependent manner. We also show that spermidine and mitoxantrone compete with α1-cyt in activating TC45, suggesting that they function by disrupting the inhibitory intra-molecular bond in TCPTP. These lead compounds could be used for rational design of TC45 agonist with improved properties.Antibodies against TCPTP , EGFR phosphotyrosine 1068 , PDGFRβ phosphotyrosine 1021 (Santa Cruz), and α-tubulin (Hybridoma Bank) were used. HRP-conjugated second-stage reagents were used, as appropriate. siRNA against TCPTP has previously been shown to be specific to TCPTP and to have identical effects to another TCPTP-targeting oligo [HeLa cells (ATCC) were maintained in DMEM, 10% FBS, 2 mM L-glutamine, 5% CO2. Primary HUVECs were freshly isolated as described  and cultSerum-starved HeLa cells or mouse embryonic fibroblasts were left untreated on plastic, stimulated with 10 μM spermidine for 1 h, or in addition treated with 50 ng/ml EGF or PDGF for 5 or 15 min. Cells were lysed in Laemmli's sample buffer and resolved on SDS-PAGE gels for western blot analysis. Western blot bands were quantified using digital image analysis and only non-saturated blots were used. The bands to compare in a blot were enclosed with equal size boxes and pixel intensity was quantified using GeneGenius Bioimaging system and Genetools software (Syngene). Automatic background correction was used.96-well streptavidin plate (Costar) wells were incubated with TBS containing 2.5 μM α1-cytoplasmic tail (biotin-WKIGFFKRPLKKKMEK) or buffer alone at +4°C for 3 h. Non-specific binding was blocked with 2% BSA/TBS-Tween containing 10% FBS overnight at +4°C. Purified recombinant TC45  was incubated in phosphatase reaction buffer  in the presence or absence of synthetic integrin cytoplasmic tail peptides or the novel small molecule TCPTP activators as indicated. The samples were assayed for phosphatase activity in triplicate using DiFMUP  as a substrate. Sample with only reaction buffer and DiFMUP (blank) was used as a control.Sprouting assays from spheroids were performed as previously described , and basFive small molecule libraries were screened for TCPTP activators. The library compounds were transferred to 384-well assay plates with a Microlab Star automated liquid handling workstation (Hamilton Robotics) fitted with 100-nl 96-channel pintool . HTS mode TCPTP dispensation was carried out using a Multidrop Combi dispenser , and the plates were incubated for 10 min. at RT. Background fluorescence was measured during the TCPTP incubation. DiFMUP was added using Multidrop and the plates were incubated for 10 min. at RT. Urea was added to stop the reactions and fluorescence was measured with EnVision 2101 multilabel plate reader . Screening data was analysed and the results normalised using the B-score method, which decreases assay and instrumentation specific variation between datapoints  implemented in R, a software environment for statistical computing. (R Development Core Team 2008). B-score hits were compared with background measurement results. Hits in wells having high background signal were discarded as false positive.2. Cell Titer Blue was added to wells and incubated for a further 4 h after which fluorescence at 560 nm was measured with Envision. Data analysis was performed using GraphPad Prism software .TCPTP wt and knockout mouse embryonic fibroblasts were applied on 384-wells, 1500 cells in 35 μl medium  per well. Seven different compound dilutions were prepared in DMSO of Spermidine, Mitoxantrone, Ruthenium Red and MDL. Compounds were added in medium to wells, with 6 replicates of each dilution. The resulting end concentrations of the compounds in the wells were 100 nM, 1 μM, 10 μM, 30 μM, 100 μM, 300 μM and 1 mM. Sample containing only DMSO was used as a negative control. Plates were incubated for 72 h at 37°C, 5% COStatistical analyses were performed using the two-tailed Student's t-test. All results are expressed as the mean ± SEM. Following P-values were used to show statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.15H13N5) and F12 (C30H38N4O2), the chemical structures of which are shown in Fig. We performed a high-throughput screen to identify small molecule compounds capable of activating TC45. Five small molecule libraries, Microsource Spectrum with 2000 biologically active and structurally diverse compounds, LOPAC1280 with 1280 pharmaceutically active compounds, Tripos with 6000 compounds, ChemDiv with 25000 compounds and ChemBridge with 30000 compounds were screened in an in vitro phosphatase assay. The assay was based on incubation of purified recombinant TC45 with a fluorescent phosphatase substrate in the presence or absence of small molecule compounds. The compounds were printed on 384-well plates. Compound interference was taken into account by measuring background fluorescence from plates which contained the compounds and the reaction mix, but no TC45. TC45 activity assay was initiated by adding purified phosphatase to the wells and allowing dephosphorylation of phosphatase substrate DiFMUP to proceed for 10 minutes. The dephosphorylation reaction was stopped with urea and the fluorescence measured using a multilabel plate reader Fig. . Due to More detailed information on the molecular mechanism governing activation of TC45 is needed to facilitate rational drug design of activating molecules. Thus, we investigated the mechanism behind the small molecule -mediated TC45 activation. Based on our previous results, integrin α1 cytoplasmic tail residues 1164-1179 interact with the amino-terminal part of TC45 activating it by alleviating the proposed autoregulatory interaction between the C- and N-terminus of the protein [The majority of TCPTP activators identified here are previously described molecules with known cellular targets other than TC45. Therefore, it was important to characterize whether they influence cell behaviour in a TC45-dependent manner. TCPTP is a known negative regulator of many mitogenic signalling pathways and acute silencing of TCPTP induces cell proliferation in cancer cells and alterations in signalling pathways ,10,28,29TC45 negatively and site-selectively regulates PDGFRβ phosphorylation . To invePreviously we have shown that collagen-induced activation of TC45 by integrin α1β1 attenuates EGFR phosphorylation at multiple sites . Since oRecently we showed that VEGFR2 is under the negative regulation of TC45 and α1-integrin in endothelial cells . We demonstrated that activation of TC45 with cell-membrane permeable α1-TAT peptide significantly reduced the length of the VEGF-induced sprouts in three-dimensional cultures of HUVECs in a TC45-dependent manner . Here weAt present, massive efforts are being undertaken to generate drugs  that inhibit RTKs playing a central role in human diseases like cancer. Many of the targeted specific therapies have failed and a growing view is that compounds hitting several targets at the same time may be more beneficial than single specificity agents (Fojo 2008). Furthermore, many patients develop resistance to the clinically used inhibitors, which could be circumvented by targeting the same pathway via other means. TCPTP is a well-characterized negative regulator of several cancer relevant RTKs including EGFR , cMet 55, PDGFRβThe chemical structures of the small molecule activators of TC45 are presented in Fig. Spermidine is a long, flexible, positively charged polyamine involved in cellular metabolism . The facTo the best of our knowledge similar PTP agonist screens have not been published and thus the hits add to the few examples of PTP activating molecules described thus far ,40. In cThe authors declare that they have no competing interests.EM carried out the phosphatase assays, western blots, sprouting assays and also drafted the manuscript. HM and NS carried out the HTS and the secondary screening. PK carried out the data normalization and bioinformatics. PH helped with the proliferation assays, ST carried out the ELISA assays, MP co-ordinated and designed the HTS. JI designed the study, analysed the results and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/7/prepub


The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells in a pediatric population.Elevated serum immunoglobulin (Ig) E is a diagnostic marker of immediate-type allergic reactions. We hypothesize that serum IgE does not necessarily reflect total body IgE because Whole blood samples from 48 children  were analyzed by flow cytometry for FcεRI, CD23, and cell-bound IgE on dendritic cells (CD11c+MHC class II+), monocytes (CD14+), basophils (CD123+MHC class II-) and neutrophils (myeloperoxidase+). Total serum IgE was measured by ELISA and converted into z-units to account for age-dependent normal ranges. Correlations were calculated using Spearman rank correlation test.Dendritic cells, monocytes, basophils, and neutrophils expressed the high affinity IgE-receptor FcεRI. Dendritic cells and monocytes also expressed the low affinity receptor CD23. The majority of IgE-receptor positive cells carried IgE on their surface. Expression of both IgE receptors was tightly correlated with cell-bound IgE. In general, cell-bound IgE on FcεRI+ cells correlated well with serum IgE. However, some patients carried high amounts of cell-bound IgE despite low total serum IgE levels.In pediatric patients, levels of age-adjusted serum IgE, cell-bound IgE, and FcεRI correlate. Even in the absence of elevated levels of serum IgE, cell-bound IgE can be detected on peripheral blood cells in a subgroup of patients. The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells of pediatric patients.Serum IgE is an essential laboratory parameter in the diagnosis of allergies. Serum IgE levels generally correlate with the severity of allergic diseases This analysis was performed as a substudy within an ongoing prospective cohort study on the role of FcεRI in the gastrointestinal tract. Patients scheduled for an elective esophago-gastro-duodenoscopy at the Division of Gastroenterology at Children's Hospital Boston were randomly invited to participate. Subjects between 1 and 18 years of age were enrolled. Patients' characteristics such as age, gender, and race were obtained in a structured interview. Subjects with a recent (<3 months) use of steroid in any form, immunomodulatory drugs, mast cell stabilizer, or leukotriene inhibitor, as well as patients with an established diagnosis of autoimmune, inflammatory, or immunodeficiency disease and patients under 1 year of age were excluded from the study. The study protocol was approved by the Investigational Review Board at Children's Hospital Boston . Patients or their legal guardians provided written informed consent. We obtained peripheral blood samples for serum IgE measurements and flow cytometry analysis at the time of enrollment.2SO4 before reading ODs at 450 nm. Normal serum IgE levels are given by the manufacturer as <10 IU/ml for age 0–3 years, <25 IU/ml for 3–4 years, <50 IU/ml for 4–7 years, <100 IU/ml for 7–14 years, and <150 IU/mL for adults older than age 14. IgE levels above the age specific normal range are referred to as ‘elevated’ IgE from here on.Total serum IgE was determined using a solid-phase ELISA  according to the manufacturer's instructions. In brief, plates pre-coated with an anti-IgE monoclonal antibody were incubated with patient sera and a second anti-IgE antibody conjugated to horseradish peroxidase. After washing, levels of bound IgE were determined by incubating with tetramethylbenzidine (TMB) solution and stopping the reaction with 2N Hhttp://www.bdbiosciences.ca/canada/downloads/is_protocols/IF_WB_LW.pdf). The following antibodies were used: anti-IgE FITC (Invitrogen), anti-FcεRIα APC , anti-CD23 APC, anti-myeloperoxidase (MPO)-PE for neutrophils, anti-CD14 PerCP-Cy5.5 for monocytes, anti-CD11c V450 and anti-MHC class II-PerCP  for dendritic cells. Basophils were identified by anti-CD123-PE (BD) and negativity for MHC class II.100 microl of K2EDTA blood were stained following the Becton Dickinson protocol for direct immunofluorescence staining of whole blood . After surface staining, neutrophils were identified by intracellular expression of MPO using FIX&PERM®  according to the manufacturer's guidelines. Cells were washed and resuspended in PBS for flow cytometry analysis. Corresponding isotype controls were performed for each staining.Data were acquired with a DakoCytomation MoFlo Legacy  and analyzed with Summit for Windows Version 4.3 (Dako North America Inc.). Typically, 20,000 events within the granulocytes/monocytes gate in the forward/side scatter plot were acquired. FcεRI and CD23 levels were determined as percent (%) positive cells among cell type after subtraction of the individual isotype control background. Cell-bound IgE levels were recorded as the percentage of cells that were double positive for IgE and FcεR1 or IgE and CD23 after subtraction of background.Data were analysed with SPSS for Windows . Chi-square test was used for nominal variables. Continuous variables were compared by Student's T-test or Mann-Whitney-U where called for. Correlations were calculated using Spearman's rank correlation test since datasets were not normally distributed. Spearman's rank correlation coefficients are displayed as ‘rho’. To account for age-dependent normal ranges, total IgE levels were converted into z-units as published by Kjellman et al. A total of 48 pediatric patients were included in the study between October 3, 2008 and July 17, 2009 . The characteristics of the study population are given in All patients expressed FcεRI on their dendritic cells (DCs). FcεRI-expression ranged between 2.54 and 26.43% FcεRI+ DCs . The majPatients with elevated serum IgE (n = 14) showed a significant correlation between age-normalized serum IgE z-units and FcεRI on dendritic cells (rho = 0.552, p = 0.041, Numbers of CD23+ cells were lower than FcεRI+ cells . DCs froFcεRI+ DCs from 45 of 48, FcεRI+ monocytes and basophils from 41 of 48, as well as FcεRI+ neutrophils from 28 of 47 patients stained positive for IgE . PercentOn FcεRI+ DCs, monocytes, and neutrophils, a positive correlation between cell-bound IgE and absolute total serum IgE was detected IU/l, . AccordiCell-bound IgE was linked to IgE-receptor expression. FcεRI-expression significantly correlated with cell-bound IgE in FcεRI+ DC's, monocytes, and basophils. CD23-expression correlated with cell-surface IgE in CD23+ DC and monocytes .We next analyzed whether elevated serum IgE is reflected by high numbers of cell-bound IgE. We found that numbers of IgE-positive cells did not significantly differ between children with elevated and normal serum IgE (Mann-Whitney-U test, In this work we have correlated levels of cell-bound IgE on peripheral blood cells of pediatric patients with total serum IgE and IgE-receptors. Analyzing expression of the high affinity IgE receptor FcεRI, we observed a slight increase of FcεRI expression on DC and monocytes with age, which is in line with previous findings by Wada et al. An association between cell surface density of FcεRI and serum IgE levels has been described for adult atopic patients CD23 expression was not associated with serum IgE, which is consistent with a previous report In our study, cell-bound IgE on FcεRI+ cells generally correlated well with elevated serum IgE. In a subgroup of patients though, monocytes, basophils, and neutrophils were loaded with IgE despite normal or low serum IgE levels. Allergic diseases in the absence of elevated serum IgE levels have been described in the literature Comparable to the increase in FcεRI expression by elevated serum IgE, monoclonal anti-IgE antibodies, such as Omalizumab, decrease serum IgE and FcεRI-surface expression on dendritic cells, monocytes, and basophils In summary, we find that in pediatric patients, levels of age-adjusted serum IgE and IgE-receptors correlate. The majority of FcεRI+ cells also carry IgE on their surface. Cell-bound IgE on FcεRI+ cells correlated well with high levels of serum IgE. We also identified pediatric patients who carried IgE at the surface of peripheral blood cells while serum IgE levels were normal. Further prospective cohort studies are needed for a detailed analysis of the correlation between cell-bound IgE with clinical symptoms of allergy and therapeutic responses. We propose here that measuring cell-bound IgE could be a valuable additional laboratory parameter in the assessment of IgE-mediated allergies.


Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as  Yersinia pestis, the plague bacterium, which injects its effector proteins into the host cells by the type III secretion system Yersinia effector (Yop) proteins include the eukaryotic-like serine/threonine kinase YpkA, also known as YopO Despite their clinical relevance, the mechanisms employed by pathogenic bacteria to subvert the host immune system remain only partially characterised. It has become clear, however, that pathogens create a beneficial environment for their survival by secreting proteins that mimic the functions of several host proteins. One of the best known bacterial examples is Yersinia genus, but they have also been identified in the soil microorganism Myxococcus xanthusMycobacterium tuberculosis, which even encodes 11 STPKs. Only two of these (PknG and PknK) are soluble proteins, while the other nine STPKs contain a transmembrane domain Pseudomonas aeruginosaStreptococcus pneumoniaeStaphylococcus aureusEukaryotic-like serine/threonine protein kinases (STPKs) are present not only in the S. aureus. This Gram-positive bacterium is part of the human microbiota, but it can turn into a dangerous pathogen, causing a wide range of infections S. aureus is mostly considered as an extracellular pathogen, it can invade a variety of mammalian non-professional cells, such as nasal endothelial cells. Moreover S. aureus survives phagocytosis by professional phagocytes S. aureus has evolved various protective and offensive responses Recently, attention has been focussed on the PknB kinase of S. aureusS. aureus. A direct role of PknB in infection is conceivable since serine/threonine kinases play key roles in mammalian cell signalling, and at least two bacterial equivalents, YpkA of Yersinia and PknG of Mycobacterium tuberculosis, have been shown to be directly involved in the subversion of host cells during the respective infectious processes The PknB kinase is composed of three extracellular PASTA domains (penicillin binding domains), a central transmembrane domain and an intracellular kinase domain S. aureus cells. We therefore investigated whether PknB of S. aureus can recognize and phosphorylate known substrates of human serine/threonine kinases. For this purpose, we used peptide microarrays with known human phosphorylation sites. The phosphorylation profile and mass spectrometry results show that PknB is a proline-directed kinase, which can indeed phosphorylate specific human targets. The observed target specificity of PknB indicates possible roles for this enzyme in a wide range of host cell signalling processes during S. aureus infection.Here we show that full-size soluble PknB is present in the medium of growing M. tuberculosis and Yersinia species can secrete their eukaryotic-like serine/threonine kinases directly into the host. This mechanism allows these bacteria to survive intracellularly S. aureus. As shown by Western blotting using polyclonal antibodies against PknB, the full-size PknB was detectable both in the cellular and growth medium fractions of cells of S. aureus NCTC 8325 harvested at an OD600 of 2. As expected, PknB was neither detectable in cellular nor growth medium fractions of the ΔpknB mutant. The precise mechanism by which PknB is liberated from the wild-type cells remains to be elucidated. However, there is precedence for the release of membrane proteins, or fragments thereof, into the extracellular milieu of Gram-positive bacteria, such as S. aureus, by as yet unknown mechanisms It has previously been reported that different bacteria such as S. aureus, its release may impact on human host cell functions. This could be the case upon internalization of S. aureus. Although S. aureus is primarily an extracellular pathogen, there is strong evidence that it can be internalized by a wide range of human host cells. For example, S. aureus invades non-professional phagocytes by a mechanism which requires a specific interaction between the bacterial fibronectin-binding protein and the host cell S. aureus is able to escape from the host endosome, and this fact opens up the possibility of direct interactions of released PknB with proteins of the human host Irrespective of the mechanism by which PknB is released into the extracellular milieu of 33P-γ] ATP, radioactivity was efficiently incorporated in a particular subset of the peptides on the chip. In contrast, little radioactivity was incorporated when the arrays were incubated with [33P-γ] ATP in the absence of PknB. We identified 68 potential substrates for PknB, of which the biological functions are summarised in S. aureus cells may target signal transduction mechanisms for host cell subversion. In addition, 13 potential PknB substrates are involved in gene regulation, including transcription factors, transcription regulatory proteins and RNA binding proteins. Three potential substrates play a role in immune responses and recognition, five in transport processes, ten in cell growth and maintenance , two in cell metabolism, two in stress responses, and one in apoptosis.Peptide arrays (PepChips) have previously been used successfully to profile the activity of kinases in eukaryotic cell lysates 64IVADQTPTPTR74) is derived from the Activating Transcription Factor-2 (ATF-2)  see . ATF-2 bin vitro and performed mass spectrometric analyses. As a positive control we incubated ATF-2 with p38. The results show that PknB can indeed phosphorylate ATF-2   56P peptide, the release of PknB from invasive S. aureus cells might have similar effects as its phosphorylation by JNK.Another peptide that was very efficiently phosphorylated by PknB belongs to the Bcl-2 interacting protein Bim see . In vivose (JNK)  [64], [6 by PknB . ExposurYersinia into human host cells, thereby affecting cytoskeleton integrity S. aureus and other bacteria on host cell apoptosis A third intriguing target of PknB identified through phosphorylation profiling is the cytoskeleton-associated protein paxillin. This protein has previously been reported as a target for the phosphatase YopH, which is injected by S. aureus (data not shown). These include amino acid sequences in proteins that are known to be phosphorylated by PknB, such as triosephosphate isomerase, DnaK, elongation factors, ribosomal proteins and trigger factor Interestingly, a comparison of the identified human phosphorylation sites of PknB with staphylococcal proteins revealed about 300 putative PknB target sequences in proteins of in vitro, which implies that PknB has a MAP kinase-like enzymatic activity. This view is supported by the observation that PknB is also involved in the regulation of important cellular functions in S. aureus, including central metabolic pathways in vivo, for example upon internalization of S. aureus by macrophages.To determine which amino acids are preferably phosphorylated by PknB, we generated a sequence logo based on the 15% best-phosphorylated peptides . The mosS. aureus NCTC 8325 or a pknB mutant of this strain 600 readings. Samples of 3 ml were harvested at OD600 2 and cells were separated from the growth medium by centrifugation . Bacterial cells were washed in PBS, resuspended in sample buffer  and disrupted using a Precellys 24 bead beater . Proteins from the growth medium fraction were collected by TCA-acetone precipitation E. coli DH5α cells were carried out as previously described S. aureus RN4220 trxA gene on this genomic DNA was PCR-amplified using the primers GGGGGCATATGGCAATCGTAAAAGTAA and GGGGGCTCGAGTAAATGTTTATCTAAAACTTC. The PCR product was cloned into HincII (HindII) restricted pUC18 trxA gene was excised from this plasmid with NdeI and XhoI, and ligated into the same restriction sites of pET26b(+) , downstream of the T7 promoter and upstream of an in-frame His(6)tag sequence. The resulting pET26-SatrxA plasmid was checked by sequencing and used to transform E. coli BL21(DE3) (Invitrogen) for high-level trxA expression and purification. 10 ml of an overnight culture of this strain was used to inoculate 1 liter fresh LB medium and grown until an OD600 of 0.7 was reached. Then, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM to induce TrxA production. After 3 hrs of induction, cells were harvested by centrifugation and resuspended in binding buffer  glycerol, 5 mM imidazole, 3 mM DTT, pH 7.4). Next, cells were disrupted by two passages through a French Press (2500 PSI). Cellular debris was removed by centrifugation , and the clarified supernatant fraction was applied to a nickel-charged IMAC column . Unbound sample was washed from the column with binding buffer using an ÄKTA explorer . Next, the His-tagged TrxA protein was eluted from the column using binding buffer with a gradient of increasing imidazole concentrations (up to 500 mM imidazole). The eluted fractions were checked for the presence of pure TrxA protein using SDS-PAGE and subsequent silver-staining. Further purification was achieved by concentrating the proteins with Vivaspin columns (Vivascience) and loading them on a Superdex 75 gel filtration column (Amersham) pre-equilibrated with 20 mM NaPi, 150 mM NaCl, 10% glycerol and 3.5 mM DTT, pH 7.4. Fractions containing the purified TrxA proteins were pooled and dialyzed 3 times against 20 mM Tris-HCl, pH 7.6, with 150 mM NaCl. Specific polyclonal antibodies against the purified TrxA protein were raised in rabbits (Eurogentec).All procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of competent http://www.pepscan.com/) were incubated with 50 µl of the ser/thr kinase PknB incubation mix  for 90 min in a humidified incubator. As a negative control we used the “empty” incubation mix without PknB. After incubation the peptide arrays were washed twice in 2 M NaCl (1% Tween-20) and PBS-T. Next, the arrays were rinsed twice in demineralised water and air-dried. The dried slides were transferred to a phosphor imager plate  and exposed for 72 hours. The density of the spots was measured and analyzed with array software.PepChip™ Kinomics slides containing 976 fully annotated, disease-related kinase phosphorylation sites in triplicate .To analyze the intensity of spots and to correct for background phosphorylation, the ScanAnalyze software and grid tools were used, and the resulting data were exported to an excel sheet. Three replicate data sets were taken for further statistical analysis. To this end, the Spearman correlation coefficient was calculated for each combination of the three sets. The average and standard deviation for each peptide were determined and plotted in an amplitude-based hierarchical fashion. If only background phosphorylation is present, this amplitude-based distribution can be described by a single exponent. Thus, determining the exponent describing amplitude behavior of the 500 least phosphorylated peptides should give an adequate description of array background phosphorylation and, in practice, this was indeed the case. 125 Peptides which exhibited the incorporation of γ-http://weblogo.berkeley.edu/logo.cgi using either the preferred substrates of PknB, or known phosphorylation sequences for human kinases as available at http://www.phosphosite.org/homeAction.doSequence logos were created with the weblogo server at in vitro assay was performed. Purified staphylococcal kinase PknB (26 µg) was incubated with 50 µg of Activating Transcription Factor fusion protein  in kinase incubation mix  for 30 minutes at 37°C in waterbath. As a positive control, 0.1 µg of p38-alpha MAP kinase  was incubated with 50 µg of ATF-2. As negative controls, the following reaction mixtures were used: PknB with ATF-2 but without ATP; PknB with ATP but without ATF-2; and ATF-2 with ATP but without PknB.In order to confirm the phosphorylation of ATF-2 by PknB, the S. aureus NCTC 8325 proteins and added ATF-2 protein (extracted from the NCBI database) using Sorcerer™-SEQUEST® . Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. Up to two missed tryptic cleavages were allowed. Methionine oxidation (+15.99492 Da), Carbamidomethylation (+57.021465 Da) and phosphorylation of STY (+79.966331 Da) was set as variable modification. Proteins were identified by at least two peptides applying a stringent SEQUEST filter. Sequest identifications required at least deltaCn scores of greater than 0.10 and XCorr scores of greater than 1.9, 2.2, 3.75 and 3.75 for singly, doubly, triply and quadruply charged peptides. Phosphorylated peptides which passed this filter were examined manually and accepted only, when b- or y- ions confirmed the phosphorylation site.Trypsin (Promega) was activated by 15 min incubation at 30°C in activation buffer and then added 1∶200 to the samples, digestion was allowed to proceed over night at 37°C. The resulting peptides were separated by liquid chromatography and measured online by ESI-mass spectrometry using a nanoACQUITY UPLC™ system  coupled to an LTQ Orbitrap™ mass spectrometer . Peptides were desalted onto a trap column . Elution was performed onto an analytical column  by a binary gradient of buffer A (0.1% (v/v) acetic acid) and B (100% (v/v) acetonitrile, 0.1% (v/v) acetic acid) over a period of 50 min with a flow rate of 400 nl/min. The LTQ Orbitrap was operated in data-dependent MS/MS mode using MSA for phospho-relevant masses. Proteins were identified by searching all MS/MS spectra in .dta format against all Table S1PknB-phosphorylated peptides(0.15 MB DOC)Click here for additional data file.Table S2Peptides phosphorylated by PknB grouped according to function(0.16 MB DOC)Click here for additional data file.


Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt.Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation.Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, the Gram-negative bacteria associated with periodontitis, suggests a role for PARs and particularly PAR2 as a putative mediator of periodontitis [Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) with a unique mechanism of activation. These receptors carry their own tethered ligands and are activated by proteolytic activity of serine proteases . Among tdontitis -7.Periodontitis is an infection of periodontal tissues which are the supportive structure for the teeth. In the complex structure of periodontal tissues, gingival epithelium is the first layer which encounters various periopathogens, acting as a physical barrier and playing an active role in innate immunity ,9. Oral Findings from our earlier study showed that activation of PARs induced expression of CXCL3/MIP-2b, CXCL5/ENA-78 and CCL20/MIP-3α in HOKs . CXCL3 aIn the current work, we studied differential signaling of PAR1- and PAR2-mediated innate immune responses in the induction of CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt signaling. We hypothesized that the induction of these markers by PAR1 and PAR2 is differentially mediated by activation MAPK and PI3K, and used selective inhibitors for components of these signaling pathways to study their effects on PAR signaling. The results provide a novel insight into signaling pathways involved in PAR activation.2. Fourth passage cells at 75-80% confluence were used for all experiments. Due to the possible variation between individual donors, we looked for consistent results in HOKs from at least three donors with technical duplicate for each set of experiments, unless otherwise stated.Tissue preparation and cell culture method for primary HOKs have been described previously in detail . BrieflyHuman alpha-thrombin  and recombinant human trypsin  were used to stimulate HOKs in order to activate PAR1 and PAR2, respectively. D-Phe-Pro-Arg-chloromethyl ketone dihydrochloride  and serine protease inhibitor, tosyl-L-lysine chloromethyl ketone  were used to inhibit thrombin and trypsin, respectively. Inhibitors for ERK1/2 (U0126) and its control substance (U0124), p38 (SB203580), PI3K (Wortmannin and LY294002), Akt (Akt inhibitor IV) were from Calbiochem . Rabbit polyclonal p38 MAPK (9212) and Akt (9272), and monoclonal phospho-p38 MAPK , phospho-Akt , p44/p42 MAPK , and phospho-p44/p42 MAPK  antibodies were obtained from Cell Signaling Technology, Inc. . Mouse monoclonal GAPDH, used as western blot control for equal gel load of protein, was purchased from Santa Cruz Biotechnology Inc. . As assessed by the Limulus Amebocyte Lysate Pyrotell , the thrombin (1 U/ml) and trypsin (1 nM) preparations in our study contained less than 0.03 EU LPS/ml.Single-stranded cDNA was synthesized from total RNA and used to perform QRT- PCR with gene-specific primers as described previously ,12. UnstSecreted CXCL5 and CCL20 were measured in culture supernatant using Duoset ELISA kit . The absolute concentration in each sample was calculated based on sample yielding optical density using the standard curve. Chemokine secretion from each condition was normalized to chemokine produced at baseline level in unstimulated control group, and results are presented as percent of unstimulated control cells.To assess phosphorylation of p38 and ERK1/2, treated cells were homogenized in lysis buffer in accordance with the manufacturer's protocol (R&D). Immediately prior to adding to cells, protease and phosphatase inhibitors, PMFS and sodium orthovanadate (Santa Cruz Biotechnology), Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail , were added to the lysis buffer. Phosphorylation of p38 and p42/44 (ERK1/2) was assessed in cell lysate using Duoset antibody pairs following manufacturer's protocol (R&D). Total p38 was quantified by a similar method (R&D) and served for normalization. Total p38 was proportional to total protein measured in each sample and to the number of cells plated in each well.Total and phosphorylated kinase expression in untreated and agonist-stimulated HOK cells was determined by Western immunoblot analysis. Samples of whole cell lysate supernatant proteins, solubilized in 1X NuPage LSD sample and reducing buffer, were resolved concomitantly with Precision Plus Dual Color protein molecular weight standard  in NuPAGE 4-12% Bis-Tris mini gel  and MOPS SDS running buffer in XCell SureLock Mini-Cell, transferred to PVDF membrane  in XCell II Blot Module using electrophoresis systems and protocols of InVitrogen Corp. . PVDF membranes were blocked for 1 h with 1% BSA, 1%PVP and 1% PEG in wash buffer , then: (1) incubated with rabbit primary antibodies either overnight at 4°C for phospho-Akt antibody, or 1 h at room temperature for total  and phospho- proteins  at concentrations recommended by the manufacturer, then incubated for 1 h with donkey anti-rabbit IgG (H+L) HRP conjugate  diluted 1:20,000 in 0.1% Tween-20, Tris Buffered Saline (TBS); or (2) incubated with mouse monoclonal GAPDH diluted 1:5,000 in blocking buffer and then with goat anti-mouse IgG (H+L) HRP conjugate (JacksonImmunoResearch) diluted 1:50,000 in 0.1%TTBS. Secondary antibody binding to primary antibody was detected using SuperSignal West Dura Extended Duration chemiluminescent substrate and protocols of Thermo Scientific . Film exposures were chosen to give unsaturated blot densities. GAPDH immunoblots showing equal gel load of protein were obtained without stripping. Total protein immunoblots for Akt, p38 MAPK and p44/p42 MAPK were obtained from separate gels loaded with 2 to 5 fold less protein. Chemiluminograms of immunoblots were digitally scanned and saved in TIFF format using Photoshop™ software  without further processing.Data collected from at least three donors are presented as mean ± standard error of the mean (S.E.M). Data were compared using one-way ANOVA followed by Bonferroni post test. A p-value < 0.05 was considered to be significant.We examined the dynamic activation of ERK1/2 and p38 when cells were stimulated with thrombin and trypsin. ELISA-based analysis showed a transient weak and rapid (5 min) phosphorylation of ERK1/2 subsequent to both PAR1 and PAR2 activation. In PAR1-activated cells, the level of ERK1/2 activity was at its maximum within 5 min and then decreased and remained at a steady level close to baseline up to 90 min. In contrast, after its maximum phosphorylation at 5 min, PAR2 activation induced dephosphorylation of ERK1/2 to below the baseline level for up to 90 min Figure . Both PAIn our previous studies we found that thrombin induced CXCL3 and CXCL5 via PAR1, while trypsin induced up-regulation of CXCL3, CXCL5 and CCL20 via PAR2 activation . In thisThe PI3K/Akt signaling pathway plays a role in coordinating defense mechanisms in innate immunity ,19. IncrA previous study reported inhibitory effect of PI3K signaling pathways following activation of Toll-like receptor 4 by LPS . In our Since activation of MAPK and PI3K signal transduction had opposite effects on innate immune responses induced by PAR activation in HOKs, we hypothesized that PI3K has inhibitory effect on activation of ERK1/2 and p38 downstream of PAR1 and PAR2 signaling. We assayed the phosphorylation of ERK1/2 at 5 min and p38 at 30 min (the time of maximum phosphorylation of ERK1/2 or p38) when PI3K activity was inhibited by Wortmannin at various concentration and cells were stimulated with thrombin or trypsin for PAR activation. ELISA-based assay suggested that in these conditions, inhibition of PI3K by Wortmannin followed by PAR1 or PAR2 activation caused decreased phosphorylation of ERK1/2 in a dose-dependent manner Figure . In contThe transmission of signals from cell membrane into the nucleus requires coordinated action of diverse signaling proteins. In this study we identified the key signaling molecules involved in the induction of innate immunity in human oral keratinocytes in response to PAR1 and PAR2 activation. PAR1 and PAR2 have been demonstrated to activate members of the MAPK signaling cascade in the induction of IL-8 and IL-1b in epithelial cells from different tissue origin -24. In aOur kinetics studies demonstrated transient ERK1/2 phosphorylation by PAR1 and PAR2 activation, which was followed by a distinct pattern of ERK1/2 dephosphorylation. This was more prominent with PAR2 activation compared to PAR1 activation. This may explain the minimum effect of inhibition of ERK1/2 for PAR2-mediated innate immune responses. On the other hand, PAR2 activation, compared to PAR1, resulted in more effective phosphorylation of p38. These data suggest that dephosphorylation of ERK1/2 following PAR2 activation may be a protective mechanism against excess innate immune responses via p38 and ERK1/2. A similar protective effect by down-regulation of MAPK signaling downstream of PAR2 activation is reported in acute pancreatitis induced by an intraperitoneal injection of caerulein in rats . HoweverInhibition of p38 also differentially affected the expression of selected markers induced by PAR1 and PAR2 activation with different sensitivity to the presence of inhibitor for each marker. This may be related to the involvement of different p38 subunits  with differential downstream signaling and also to the lack of the equipotency of the current inhibitor against all subunits .et al. showed that PI3K/Akt is involved in modulation of NF-κB and expression of ICAM-1 induced by thrombin in endothelial cells. Their study suggested that activation of PI3K/Akt leads to activation of mTOR. While the over-expression of the catalytic domain of Akt increases activation of NF-κB in the absence of mTOR activity, restoring mTOR signaling dampens activation of NF-κB and induction of ICAM-1 [Our studies suggest PI3K has an inhibitory effect on PAR signaling in HOKs. This effect was shown most clearly at the mRNA level and also for CXCL5 at protein level. We did not observe this effect in the secretion of CCL20, which may be related either to the peptide structure of CCL20 which is vulnerable to proteolytic activity of enzymes, or to involvement of other mechanisms that affect CCL20 expression at the post-transcriptional level. Little information is available about PAR-mediated PI3K signaling in normal human keratinocytes with comparable cellular function, but our results indicate HOKs have a unique signaling system. It has been shown that thrombin signals via PI3K to induce osteoprotegerin in human periodontal ligament and VEGF in human pigment retinal epithelial cells ,27. In af ICAM-1 . In an ef ICAM-1 . In contf ICAM-1 -32, suggOur results suggest that the mechanism of crosstalk between PI3K and PAR signaling is via effect on phosphorylation of p38 and ERK1/2. We observed inhibition of PI3K resulted in increased p38 phosphorylation even in the absence of external stimulants (thrombin and tryspin), and this effect was significantly greater when cells were stimulated with active enzyme versus inactive form of thrombin and trypsin. This finding suggested a specific role of PAR activation in the induction of a crosstalk between PI3K and p38. This interaction between p38 and PI3K signaling pathways downstream of PARs activation may serve as a protective strategy HOKs utilize to keep innate immune responses in balance. Activation of PI3K inhibits the induction of proinflammatory chemokines possibly by suppression of p38 MAPK activation. When TLR5 is activated by flagellin in intestinal cells  and in VOur studies suggested that PAR1 signals via both ERK1/2 and p38, but that ERK1/2 has a more prominent role. However, there was no role for either p38 or ERK1/2 in the induction of CCL20 by PAR1 activation, although its expression was increased when PI3K and Akt were inhibited. It is likely that an alternative mechanism, which is independent of the ERK1/2 and p38 pathways, but still blocked by PI3K, is involved in the induction of CCL20 by PAR1 activation. This is consistent with our previous study showing that CCL20 induction by thrombin may occur via a mechanism other than PAR1 .Induction of cytokines and chemokines by PAR activation leads to infiltration of mononuclear cells in the microenvironment of periodontal tissue . This prWe demonstrated in this study that the induction of inflammatory responses by PAR1 and PAR2 is differentially regulated by ERK1/2 and p38 MAPK signaling pathways. ERK1/2 and p38 are both involved in signaling via PAR1, but p38 is more critical for signaling via PAR2. PI3K has a negative regulatory role limiting proinflammatory gene expression induced by both PAR1 and PAR2. We characterized crosstalk between PI3K/Akt and MAPK signaling pathways and the possibility of p38 phosphorylation as one of the mechanisms by which PI3K keeps innate immune responses in balance subsequent to PAR activation. A simple schematic overview of PAR signaling is summarized in Figure PAR: protease-activated receptor; HOK: human oral keratinocyte; PPACK: D-Phe-Pro-Arg-chloromethyl ketone; TLCK: tosyl-L-lysine chloromethyl ketone;MGR designed experiments, performed QRT-PCR and ELISA, and drafted the manuscript. DHD and JYA stimulated cells and conducted Western blot analyses. BMH performed GECs culture. BAD oversaw experimental design and helped drafting of the manuscript and discussion. WOC oversaw experimental design, contributed to discussion, drafting and revision of the manuscript. All authors read and approved the final manuscript.


The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin. Escherichia coli produce the plasmid-encoded cytotoxin colicin E3. It is a ∼60 kDa protein with three domains that are required for the toxin to enter and kill the cell. The receptor binding domain binds to the outer membrane protein, BtuB, of competing E. coli  domain specifically cleaves the phosphodiester bond between A1493 and G1494 of 16S rRNA of the small ribosomal subunit (30S) and abolishes protein synthesis . The cleEnterobacter cloacae and has a very similar mode of action as colicin E3, has been studied in more detail. It was reported that cloacin DF13-treated ribosomes were able to form initiation complexes, but the function of translation initiation factor 1 (IF1) was altered because it could no longer increase the rate of exchange between ribosome subunits or the rate and extent of IF3-mediated dissociation of 70S ribosomes  (3H]fMet-tRNAfMet in the P site were used (data not shown). In all following experiments, equimolar concentrations of colicin E3 and 70S ribosomes were used  . The trecedures) ; accordiColicin E3-treated ribosomes were tested in a translation assay reconstituted from purified components , using aTo identify the step that is impaired by the cleavage of 16S rRNA, colicin E3-treated ribosomes were tested for their ability to perform factor-dependent initiation  Caloger, indicat14C]Phe-tRNAPhe, and the amount of [14C]Phe-tRNAPhe bound to the A site was determined by nitrocellulose filtration ·GTP·[ltration . Controle A site . For furKd) of the codon–recognition complex can be measured. Titration of 70S initiation complexes with EF-Tu(H84A)·GTP·[14C]Phe-tRNAPhe gives Kd = 3 ± 0.4 nM for control ribosomes, consistent with previous measurements , and the yield of dipeptide was reduced to about 70% compared with control ribosomes. It is unlikely that the peptidyl transfer reaction per se was affected by the cleavage of 16S rRNA, because the peptidyl transferase centre is located on the 50S subunit and the rate of fMet-tRNAfMet reaction with the aa-tRNA analogue puromycin (Pmn) was not altered . The decreased rate of fMetPhe formation is therefore likely to be caused by a decreased rate of Phe-tRNA accommodation in the A site, whereas the lower final level indicated drop-off of part of the Phe-tRNAPhe from the ribosome during the accommodation phase.Another effect on A-site binding was observed when the rate of fMetPhe dipeptide formation was monitored by quench-flow . Colicin14C]Phe-tRNAPhe was accommodated and peptide bond formation had taken place, the stability of the fMetPhe-tRNAPhe binding was similar on colicin E3-treated and control ribosomes  HPLC. The rate of translocation on control ribosomes was about 10 s−1 (see below); therefore, the overall rate of coupled translocation and Pmn reaction is expected to be about 5 s−1. It should be noted that a rapid Pmn reaction is indicative of full translocation of tRNAs and mRNA on both ribosomal subunits, because partially translocated peptidyl-tRNA Val or [14C]Phe were found in the pentapeptide fraction. By extrapolating these results to longer peptides, one can expect that essentially no peptides longer than about eight amino acid residues will be formed when the ribosomes are cleaved by colicin E3. The progressive decrease in the amount of ribosome-bound aa-tRNA shows that defects in A-site binding, particularly in the presence of competing ternary complexes, is the major reason for the halt of protein synthesis.The results shown so far suggest that the strongest effect of colicin E3 cleavage is on A-site binding of ternary complex or aa-tRNA, whereas the effects on initiation, peptide bond formation and translocation are either small or cannot explain the inhibition of translation. To identify the step at which translation is halted, an assay was devised to monitor the stepwise synthesis of a pentapeptide. Initiation complexes with ffMeColicin E3 rRNase was overexpressed as a complex with His-tagged immunity protein Im3. After purification of the complex by Ni-NTA affinity chromatography, colicin E3 was separated from Im3 by denaturation with 6 M guanidinium chloride where the Im3 remains bound to the Ni-NTA resin while the E3 rRNase is eluted. Isolated colicin E3 was refolded by dialysis against water, followed by dialysis against 50 mM potassium-phosphate (pH 7.0). Final purification was by gel filtration on a Superdex 75 column (Amersham-Pharmacia-Biotech) in the same buffer. Purified colicin E3 was dialysed against water, lyophilized and stored at −20°C (Ribosomes were incubated in buffer A with colicin E3 (for concentrations see text) at room temperature for 30 min. The cleavage reaction was stopped by the addition of Im3 followed by ethanol precipitation in 0.3 M sodium acetate, pH 5.2. After washing with 70% ethanol, the pellets were re-suspended in 3 M sodium acetate, pH 5.2, containing 0.5% SDS, and incubated for 10 min at 37°C. Proteins were removed by extracting the solution three times with water-saturated phenol followed by chloroform extraction. The rRNA was ethanol-precipitated, re-suspended in water and analysed by denaturing polyacrylamide gel electrophoresis.Ribosomes (1 μM) were incubated in buffer A with colicin E3 (1 μM) (E3-treated ribosomes) or without the rRNase (control ribosomes) for 30 min at room temperature. Im3 (1.1 μM) was added to each sample and incubated for another 5 min. These ribosomes were used for the biochemical assays.3H]Met-tRNAfMet (1 μM) to form initiation complexes. Ternary complex was prepared in buffer B with putrescine (6.4 mM), phosphoenolpyruvate (3 mM), GTP (1 mM) and EF-Tu (50 μM) and incubated for 15 min at 37°C. Total aa-tRNA (21 μM) containing [14C]Leu-tRNALeu was added to EF-Tu, and the mixture was added to initiated ribosomes. After incubation at 37°C for various times, samples were removed and the amount of protein synthesized was determined by TCA precipitation and quantifying radioactivity.Total translation was assayed as described (3H]fMet-tRNAfMet (1.5 μM), mRNA (1.5 μM), GTP (1 mM) and initiation factors (1.5 μM each) and incubating for 1 h at 37°C. EF-Tu·GTP was prepared by adding GTP (1 mM), phosphenolpyruvate (3 mM) and pyruvate kinase (0.1 mg ml−1) to EF-Tu (0.9 μM) and incubating for 15 min at 37°C. Ternary complex was formed by the addition of [14C]Phe-tRNAPhe (0.8 μM) and incubation for a further minute. Ternary complex  was added to initiation complexes (0.2 μM) and incubated at 37°C. At the indicated times, samples of ribosome complexes (10 pmol) were removed for analysis. The GTPase activity of EF-G and translocation under turnover conditions were measured as described (Initiation complexes were formed by mixing 70S ribosomes (1 μM), Val–tRNAVal·GTP·EF-Tu, Tyr-tRNATyr·GTP·EF-Tu, Ile-tRNAIle·GTP·EF-Tu, Phe-tRNAPhe·GTP·EF-Tu (1.1 μM each), EF-G (2 μM) and GTP (1 mM) were added; the reaction was incubated for 10 min at 37°C. A total of 10 pmol of ribosome complex was removed and analysed by nitrocellulose filtration or HPLC peptide analysis. The experiments were carried out in parallel with 14C-labelled Tyr, Ile or Phe instead of Val in the presence of the other four unlabelled aa-tRNAs.To synthesize pentapeptides, 70S ribosomes (1 μM) were initiated with mRNA coding for MetValTyrIlePhe (3 μM) as described above and [fMet in the P site and fMetPhe-tRNAPhe(Prf16/17) in the A site. Pre-translocation complexes (0.1 μM) were mixed rapidly with an equal volume of EF-G (2 μM after mixing) and GTP (1 mM) in buffer A at 37°C. Proflavin fluorescence was excited at 470 nm and measured after passing a cut-off filter .Fluorescence stopped-flow measurements were performed and data analysed as described previously  in buffer A were mixed in buffer A at 37°C. MDCC fluorescence was excited at 425 nm and measured after passing a cut-off filter .Pi release from EF-G and EF-Tu was measured using the fluorescence change of phosphate-binding protein labelled with MDCC (2.5 μM)  were mixed with a threefold excess of ternary complex, then the reaction was quenched by 0.8 M KOH . Samples


Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC).TIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA.RT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 μM, while that for UDP was 3.24 +/- 0.82 μM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 μM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation.Here, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets. Theca cells form a multilayer cover that surrounds the follicle beginning in its early developmental stages. The main physiological roles recognized for theca cells are the initial steps in the steroidogenic process; specifically, these cells convert acetate or cholesterol to androgens , which aSeveral studies in recent years indicate that the purinergic signaling system is functionally expressed in the ovary of several species  and reprATP is an important neurotransmitter in the peripheral nervous system , and nerq/11 proteins, and they activate phospholipase C (PLC) and consequently diacylglycerol and phosphoinositide-Ca2+ turnover; subtypes 12-14, on the other hand, are coupled to Gαi/0 proteins that signal primarily by inhibiting adenylyl cyclase i changes, suggesting that multiple P2 receptor subtypes are expressed in theca cells. Here, we studied this response in order to elucidate in more detail the molecular elements involved and the physiological implications of their activation. We found that uridine-sensitive P2Y2 and P2Y6 receptors are expressed in the TIC membrane and that P2Y activation promoted three important responses in these cells: 1) elicited Ca2+ mobilization from intracellular reservoirs, increasing the concentration of this important second messenger in the cytoplasm; 2) increased cell proliferation through a mechanism dependent on the activation of protein kinase C (PKC) as well as MAPK p44 and p42, and; 3) down regulated hCG-dependent phosphorylation of CREB, an important element in steroidogenesis cascade control.Recently, it was demonstrated that functional P2X7 receptors are expressed in mammalian TIC and can induce apoptotic cell death . In the taq polymerase, and other molecular biology reagents were purchased from Invitrogen Co. , and Fluo4-AM was from Molecular Probes Invitrogen Co. . Automatic sequencing was done in the Molecular Biology Unit of the Instituto de Neurobiología, UNAM.ATP, UTP, UDP, suramin, human chorionic gonadotropic hormone (hCG), porcine follicle stimulating hormone (FSH), and PPADS were purchased from Sigma Chemical Co. , and staurosporin, wortmanin, and phorbol 12-myristate 13-acetate (PMA) were from Calbiochem . DMEM-F12 medium, fetal bovine serum, antibiotic-antimycotic mix, and other cell culture reagents were from Gibco Invitrogen Co. . Antibodies against mouse total or phosphorylated MAPK p44 and p42 and total or phosphorylated CREB were from Cell Signaling , and antibody against poly(ADP-ribose) polymerase-1 (PARP) was from Santa Cruz Biotechnology . Oligonucleotides, reverse transcriptase, oligo dT, 2. Cultures were maintained 48 h before using them in experiments, to allow proper recovery after the isolation procedure. With this method we usually obtained 1 × 106 cells per mouse; purity was assayed by RT-PCR and immunocytochemistry against CYP11A protein (> 96% of cells were positive). To exclude contamination with granulosa, the expression of the FSH receptor transcript and the responsiveness of CREB phosphorylation to hCG or FSH were assayed.Mouse theca/interstitial cells were purified by a discontinuous Percoll gradient ,20. ImmaTotal RNA of TIC cultures or from the indicated organ was purified using the guanidine isothiocyanate method . First sβ-actin, p2y2r, p2y4r, and p2y6r transcripts, and for cyp11A, cyp17A, star, and fshr as positive and negative theca cell markers, respectively. All the PCR programs started at 96°C for 3 min and finished at 72°C for 1 min. The amplification cycles consisted in 40 s at 96°C, 40 s at the specific annealing temperature for each primer set, and 40 s at 72°C.The cDNA was used as template in a polymerase chain reaction to amplify cDNA fragments for p2y2r, forward GGACGAACTGGGATACAAGTGT, reverse GTGGACTCTGTCCGTCTTGAGT, annealing temperature 55°C, 30 cycles; p2y4r, forward GGGACTAACTGCAGGCAGAG, reverse GATACACATCAGGCCCGTCT, annealing temperature 60°C, 40 cycles; p2y6r, forward TTTCAAGCGACTGCTGCTAA, reverse TGGCATAGAAGAGGAAGCGT, annealing temperature 55°C, 30 cycles; cyp11A, forward GCTGGAAGGTGTAGCTCAGG, reverse CACTGGTGTGGAACATCTGG, annealing temperature 55°C, 30 cycles; cyp17A, forward TGGTCGGCCCCAGATGGTGA, reverse ATCTCGGGACTCCCCGTCGT, annealing temperature 56°C, 30 cycles; star, forward AACCAGGAAGGCTGGAAGAAG, reverse AGCAGCCAAGTGAGTTTAGTC, annealing temperature 55°C, 30 cycles; fshr, forward TGGATGTCATCACTGGCTGT, reverse CAAATCTCAGTTCAATGGCG, annealing temperature 58°C, 30 cycles; and β-actin, forward GGGTCAGAAGGATTCCTATG, reverse GGTCTCAAACATGATCTGGG, annealing temperature 55°C, 25 cycles.The sequences of the oligonucleotides, the annealing temperatures, and the number of PCR cycles used were as follows: The amplified products were gel-isolated, phenol-chloroform purified, and subcloned into the pCR4-TOPO vector (Molecular Probes Invitrogen Co.). Their nucleotide sequences were confirmed by automatic sequencing.2, 1.5 CaCl2, 4 glucose, 10 HEPES, and 0.05% BSA, pH 7.4). The cells were washed with Krebs solution for 10 min to eliminate excess dye and then placed in a constant-flow recording chamber that allowed them to be visualized with an inverted fluorescence microscope . Drugs were applied by superfusion and responses were recorded with an Evolution QEi camera . Sequences of images were analyzed using the Image-Pro Plus software  and Imagenes software, a program developed specifically for this analysis . In the Ca2+-free Krebs solution, CaCl2 was replaced by 3 mM MgCl2.Mouse ovarian TIC were grown on 12-mm diameter cover slides. Semi-confluent cultures were loaded for 15 min with 5 mM fluo-4/AM and 0.1% pluronic acid in Krebs solution  were harvested 24 h before the experiment to reduce serum-dependent kinase activity. After that, cells were stimulated with the indicated drugs, scraped in Laemmli buffer, and boiled for 5 min. For electrophoresis, samples were fractionated in a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane . Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4°C with the appropriate rabbit primary antibody (1:1000) directed against the phosphorylated form of MAPK p44 and p42 or CREB . After washing with TBS-T, membranes were incubated 1 h at 37°C with HRP-conjugated goat anti-rabbit antibody  in TBS-T. The immunoreactive proteins were detected by chemiluminescence, and images were analyzed by pixel density with ImageJ Software ; the results were expressed in terms of optic density normalized against the basal condition, a parameter that is proportional to the change in protein phosphorylation. To analyze total p44/p42 or other load controls, such as PARP, the same membranes were incubated for 30 min in striping solution  at 55°C, washed twice with TBS-T, and then reprobed with a primary antibody against the indicated protein.For MAPK p42 and p44 or CREB phosphorylation experiments, cultured TIC (5 × 106) were scraped in ice-cold TNTE buffer  containing 5% Triton X-100 and a protease inhibitor cocktail ; the lysate was centrifuged for 10 min at 14,000 rpm at 4°C, and the soluble fraction was incubated overnight with 3 μl of anti-P2Y6 antibody . After that, 50 μl of protein G agarose was added to the lysate and incubated for 1 h at room temperature; the agarose beads were washed 3 times with TNTE containing 1% Triton X-100 and protease inhibitors, resuspended in Laemmli buffer, boiled for 5 min, and analyzed by Western blot.TIC  were cultured in 48-well plates and after 48 h of culture, they were harvested and incubated for 24 h in serum-free DMEM-F12 media; then the culture medium was changed to DMEM-F12 with 0.1% fetal bovine serum containing the experimental treatment . Then cultures were incubated for another 48 h, with the addition of 1 μ Ci/well of [3H]-thymidine after the first 24 h. At the end of the incubation, each well was washed 3 times with 5% trichloroacetic acid, and then the cells were lysed by addition of 250 μl of boiling 250 mM NaOH, incubated 5 min, and transferred to vials containing 5 ml of scintillation liquid. Samples were counted in a scintillation counter.Cell proliferation was analyzed using i) using fluorescence microscopy of Fluo-4-AM loaded-cells. In all cases, 25 to 40 cells from 3 independent cultures (each from 2 donors) were analyzed , responses to either UTP or UDP were not abolished . For these experiments, UTP was used as a specific agonist of the P2Y receptor subtypes studied. It was observed that UTP induced MAPK phosphorylation in a dose-dependent manner with an ECP Figure . The timP Figure .50 of 84.3 ± 10.2 μM  transactivation of EGF receptors as has been demonstrated, for example, in salivary gland cells  or 2) byTo study the possible participation of PKC, TIC cultures were preincubated (30 min) with 250 nM staurosporine and then tested with 10 μM UTP. Staurosporine treatment blocked completely the UTP-stimulated p44 and p42 phosphorylation Figure , strongl2+ during p44 and p42 MAPK phosphorylation, cell cultures were preincubated (90 min) with 10 μM BAPTA-AM to load the cells intracellularly with this Ca2+ chelator. Fluorescence microscopy confirmed that this treatment prevented the calcium increase induced by UTP. In BAPTA-loaded TIC, phosphorylation of MAPK elicited by UTP was strongly inhibited  but not by 100 μM ATP (135 ± 13%). Also, 100 μM UTP or UDP induced robust responses of 205 ± 18% and 221 ± 17%, respectively, while at 1 mM they generated increases of 216 ± 37% and 183 ± 23%, respectively. The results strongly suggested that P2Y receptors were able to activate a proliferative response in TIC.The results indicated that ATP, UTP, or UDP increased proliferation. Incubation with 10% FBS increased ; m,32; m2,32+ signals in normal Krebs or in Ca2+-free solution, which indicated that the nucleotides promoted the response mainly through Ca2+ release from intracellular reservoirs, in agreement with the canonical Gαq-PLC pathway for these receptors ; in eceptors , glioma eceptors , and embeceptors . StaurosActivation of MAPK p44 and p42 is directly related to induction of cell proliferation . Our resATP actions to stimulate TIC proliferation through P2Y2 (and P2Y6) receptor activation should be taken into account, together with the effects described for other neurotransmitters that seem to regulate specific processes in the ovary. For example, previous evidence showed that human granulosa-luteal cells express M1 and M5 muscarinic receptors  as well The results also showed that P2Y2 receptor activation had an important effect on the LH signaling pathway. It has been shown before that LH induces CREB phosphorylation ,39 and tIs well known that during folliculogenesis LH exerts regulatory actions beginning around the formation of early secondary follicles, which is concurrent with theca layer differentiation; from this stage throughout folliculogenesis up to ovulation, LH is the main regulator of theca layer development, because it controls the steroidogenesis process . HoweverSpecifically in regulating the balance between theca proliferation and death, our data suggest that activation of the purinergic system by ATP could have dual effects on theca cell physiology; i.e., depending on the concentration, ATP might induce: 1) apoptotic cell death through P2X7 receptors 18) and 2) cell proliferation through P2Y2/P2Y6 receptors, as shown here. This is similar to what has been demonstrated in other systems in which the cells seem to co-express multiple purinergic receptor subtypes, leading to activation of multiple signaling pathways. For example, macrophages express a variety of P2X and P2Y purinergic receptors, and their activation modulates diverse physiological process such as apoptosis, activation of cell proliferation pathways, or activation of the inflammatory response machinery ,43. The  and 2) cData presented in the present work are the first evidence that UTP-sensitive P2Y receptors are expressed and functional in theca cells. Although extensive studies are necessarily to establish with detail the main physiological activities, experimental data suggested these receptors have a role in p44/p42 MAPK phosphorylation, proliferation increase, and cross talk with LH-activated pathways. These observations raise the possibility that the purinergic signaling system represents an important physiological regulator of theca cells.2+-dependent proliferative response mediated through PKC activation and phosphorylation of the p42 and p44 MAPK proteins. P2Y receptor stimulation also regulates hCG-dependent CREB phosphorylation, suggesting interactions between functional pathways. Molecular components of purinergic transmission systems represent new molecular targets that must be characterized in the context of ovarian pathophysiology.In summary, it was shown here that TIC express functional P2Y2 and P2Y6 receptors, which, when stimulated, induce a CaP2Y: Purinergic G protein-coupled receptors; MAPK: Mitogen-activated protein kinases; PPADS: pyridoxal phosphate-6-azo  tetrasodium salt; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid; CREB: cAMP response element binding protein; TIC: Theca interstitial cells; PMA: Phorbol 12-myristate 13-acetate.The authors declare that they have no competing interests.FGVC and ROA conceived and designed the study, carried out experiments, performed the data analysis, and drafted the manuscript; EPZD and GE performed experiments and participated in the analysis of data. All authors read and approved the final manuscript.