However the same concentrations of H2O2 failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFβ receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFβ receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFβ and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFβ and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type β receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H Apocynin (4'-hydroxy-3'-methoxyacetophenone) was purchased from Santa Cruz . Antibodies against β-actin, TrkB, PDGFβ receptor, and phospho-PDGFβ receptor Y1021 were also purchased from Santa Cruz. Antibodies against phospho-TrkB Y816, ERK1/2 and phospho-ERK1/2 were purchased from Cedarlane.5-HT (5-hydroxytryptamine hydrochloride), 2 at a temperature of 37°C, with media changes every 3-5 days. For experimentation, cells were plated without antibiotics, and prior to drug treatments, cells were serum starved for 24 h.SH-SY5Y cells were obtained as a generous gift from Dr. Shilpa Buch, University of Nebraska. Cultures were grown in complete growth media consisting of DMEM and Ham’s F12 in a 1:1 ratio, 10% fetal bovine serum , and penicillin/streptomycin. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2PO4, 137 mM NaCl, 0.34 mM Na2HPO4, 4.2 mM NaHCO3, 0.03 mM phenol red, pH 7.4, 320-335 mOsm/kg). The brains were removed, and the cortex was dissected and trypsinized with 0.25% trypsin for 20 min at 37°C. Cells were then strained and plated on poly-d-lysine-coated culture dishes and grown at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were plated with plating media  for the first 2-4 h until cells attached. Media were then replaced with feeding media consisting of Neurobasal medium and B-27 supplement  without serum, and half of the media volume per well was changed twice per week. Drug treatments were performed 7-8 days after plating the cells. To prevent the overgrowth of non-neuronal cells, a mitotic inhibitor (81 µM 5-fluoro-2’-deoxyuridine and 200 µM uridine added to media) was added for 24 h once cells reached confluency. All animal experiments were performed in strict accordance with the guidelines and policies on the Use of Animals at the University of Waterloo, and all efforts were made to minimize discomfort. The protocol was approved by the Waterloo Office of Research Ethics Animal Care Committee .CD-1 mouse embryos  were removed at E17 to E19 and transferred to chilled dissection media  prior to use) was added and lysates were homogenized and centrifuged at 13,000 x g for 20 min at 4°C. Supernatant protein concentration was determined using the BCA protein assay  and samples were normalized. Loading buffer  was added to samples, which were then heated for 15 min at 75°C. SDS-PAGE was used to separate proteins followed by transfer of proteins to nitrocellulose or PVDF membranes. 5% non-fat milk in Tris-buffered saline plus 0.1% Tween (TBS-T) was used to block membranes for 1 h at room temperature or overnight at 4°C. Membranes were then incubated with primary antibody for 1 h at room temperature or overnight at 4°C. Membranes were washed three times with TBS-T, and then incubated with secondary antibody conjugated to horse radish peroxidase (HRP) for 1 h at room temperature. Membranes were washed three additional times with TBS-T. Proteins were visualized with western chemiluminescent substrate  on a Kodak 4000MM Pro Imaging Station. Kodak Molecular Imaging software was used for densitometric analyses of images and data statistics were evaluated with GraphPad Prism software with statistical significance set at p < 0.05. After imaging, membranes were stripped and re-probed with other antibodies.2O2 treatments, media was changed to serum-free, phenol red-free DMEM/F12 and cultures were returned to the cell culture incubator for 24-48 h to allow mitochondrial enzyme deactivation in non-viable cells. MTT reagent -2,5-diphenyl-2H-tetrazolium bromide; Sigma) was then added to cell culture media, and plates were returned to the cell culture incubator for 2-4 h for the reaction to occur. Cells were then lysed and resulting crystals dissolved in solubilization buffer  on a gyratory plate shaker. Plates were read at 570 nm absorbance and background absorbance at 690 nm was subtracted from these values.SH-SY5Y cells were seeded at equal concentrations and grown to 90% confluency, followed by overnight serum starvation. After H2O2 can cross the cell membrane [2O2 to SH-SY5Y cells for 5 min and observed peak tyrosine 1021 phosphorylation of PDGFβ receptor at a concentration of 0.1 µM  was able to abrogate PDGFβ receptor phosphorylation, suggesting that ROS are indeed involved in 5-HT-induced PDGFβ receptors transactivation. Because H2O2 can cause cell damage and death at high concentrations, we verified that the low concentrations of H2O2 used here  were not adversely affecting cell viability. As determined by the MTT cell viability assay, we found that the cells were unaffected by H2O2 treatment after 30 min . Furthermore, in contrast to its ability to block 5-HT-induced PDGFβ and TrkB receptor phosphorylation, pretreatment with N-acetyl-l-cysteine had no effect on 5-HT-induced ERK1/2 phosphorylation  by promoting the mitogenic effects of multiple RTKs. This is in line with other studies suggesting that transactivation is cytoprotective in the short term [Since Hort term , whereasort term .2O2 [phox/NOX2 (to enhance its association with other NADPH oxidase subunits) [phox [While the signaling steps downstream of ROS remain to be confirmed, we suggest that the upstream component responsible for ROS generation in transactivation pathways is NADPH oxidase. This enzyme is a large, multi-subunit complex that produces superoxide from oxygen and a donated electron from NADPH . Superox2O2 . Althoug2O2 , NADPH o2O2 . Among t2O2 ,45. Two 2O2 ,46, whilubunits)  and p47ps) [phox . Whethers) [phox ,50, whics) [phox –53.2O2-induced increases in ERK1/2 phosphorylation, an observation that contradicts previous work showing that exogenously applied H2O2 results in ERK1/2 phosphorylation [2O2 concentrations between 0.1 and 2 mM – at least 100-fold higher than the concentrations used here. The low concentrations of H2O2 used in this study compared to other systems may not be sufficient to induce ERK1/2 phosphorylation, suggesting ROS is not required for ERK1/2 activation. This is further corroborated by the ROS scavenger N-acetyl-l-cysteine being able to block RTK phosphorylation, but not ERK1/2 phosphorylation, induced by 5-HT. Conversely, the NADPH oxidase inhibitors apocynin and diphenyleneiodonium chloride were able to inhibit ERK1/2 activation. These drugs may be preventing the assembly of the oxidase or chemically modifying the subunits [Our study failed to detect Hrylation –56. Howesubunits ,58, suggsubunits ,59, it ii-coupled 5-HT receptors. This is in line with other studies showing the dependency of transactivation on Gαi-linked GPCRs including D2-class dopamine [We also show for the first time that 5-HT is capable of transactivating TrkB receptors. Like PDGFβ receptor transactivation , TrkB trdopamine , lysophodopamine , and sphdopamine , and mayi-coupled GPCRs such as 5-HT1A [i and/or Gβγ subunits [2O2 (or the exogenous application of H2O2) leads to RTK (PDGFβ and TrkB receptors) but not ERK1/2 phosphorylation. Crosstalk between 5-HT receptors and multiple RTKs may suggest that transactivation is a global pathway responsible for mitogenic or protective effects. In addition, the idea that serotonergic stimuli can activate neurotrophic factor and neuronal growth factor receptors brings together two major hypotheses for the pathophysiology of depression. Given that monoamine and neurotrophic hypotheses both propose a dysregulation in their respective signaling pathways as causes for clinical depression [A diagram of the proposed signaling pathway is presented in s 5-HT1A . PLC is subunits , which rpression ,61, it i


Cdc55 phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2ACdc55 phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2ACdc55 regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2ACdc55 initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2ACdc55 functions affect the regulation of various MEN components, contributing to mitotic exit.Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A Cell cycle studies over the years have tried to elucidate the molecular mechanisms behind cell division, one of the most highly regulated of all cell processes, which ensures life in all organisms. Protein phosphorylation emerged as a key regulatory mechanism in the cell cycle. The highly conserved family of cyclin-dependent kinases, the Cdks, are considered the main component of the cell cycle control system. However, it has become clear that opposing phosphatases also play a key role in determining the phosphorylation state of the proteins. Cells enter mitosis when mitotic Cdk activity increases, having its pick of activity during metaphase. To exit mitosis, cells must coordinate chromosome segregation with Cdk inactivation processes involving the activation of protein phosphatases. Here we show that the phosphatase PP2A regulates the mitotic exit network (MEN) by counteracting the phosphorylation of Bfa1 and Mob1. Our findings provide new insights into the mechanism by which PP2A-Cdc55 functions affect the regulation of various MEN components that contribute to mitotic exit. The core signalling elements of the MEN, SIN and Hippo pathways are highly conserved. Therefore, studies of MEN regulation will contribute to our understanding of MEN-related pathways in other organisms. Cdc55 (type 2A protein phosphatase), Zds1, Slk19, Spo12 and Fob1, have been implicated in early anaphase Cdc14 release (reviewed in Cdc20 (anaphase-promoting complex) activation, securin is degraded by the proteasome and separase is activated, allowing sister chromatid segregation and FEAR-Cdc14 release. The protease separase, the main component of FEAR, allows the Cdk1-dependent phosphorylation of Net1 by downregulating the phosphatase PP2ACdc55Cdc55During most of the cell cycle, Cdc14 is kept inactive and sequestered in the nucleolus through binding to its inhibitor Net1 Cdc55 phosphatase acts as a functional link between FEAR and MEN, due to its action on Bfa1 and Mob1. PP2ACdc55 downregulation at anaphase onset facilitates Bfa1 inactivation and thereby initiates the MEN pathway up to the Cdc15 kinase. Premature Bfa1 inactivation observed after PP2ACdc55 inactivation does not entail precocious MEN activation, since the downstream MEN effectors Cdc15 and Dbf2–Mob1 are kept inactive by Cdk1–Clb2 phosphorylation. In this way, Cdk1–Clb2 restrains MEN activity until mid-late anaphase allowing a consecutive order of FEAR- and MEN-Cdc14 functions.Here, we propose that PP2ACdc55 could facilitate anaphase-specific phosphorylation of Bfa1 The Bub2–Bfa1 complex keeps MEN inactive by inhibiting Tem1. Phosphorylation of Bfa1 by Polo kinase, Cdc5, contributes to MEN activation in an anaphase-specific manner Cdc55 in regulating MEN activity, we studied the dephosphorylation of Bfa1 by PP2ACdc55. First, we confirmed that Bfa1 was already phosphorylated in metaphase in a cdc55Δ mutant  due to the loss of integrity of the PP2A trimeric complex in this mutant. Taken together, these results suggest that Bfa1 is likely to be an in vivo substrate of PP2ACdc55.Thirdly, we determined Bfa1 phosphorylation status under Zds1 overexpression, which promotes PP2Ativation . This inpression . Next, walactose . Bfa1 bell cycle . MoreoveCdc55 inactivation would depend on Cdc5. To test this, we compared Bfa1 electrophoretic mobility after Zds1 induction in wild-type and cdc5-14 mutant cells , and cdc55Δ mutant cells were grown in the presence of unmodified arginine and lysine (light). Cells were arrested in metaphase and protein extracts were prepared. Phosphopeptides were enriched by SIMAC-based enrichment. Phosphopeptide analysis of the heavy/light-labelled cells was done by LC-MS/MS. An already known PP2ACdc55 substrate Net1 was identified as being hyperphosphorylated in the cdc55Δ mutant, suggesting that the technique worked properly. The screening revealed that a phosphopeptide corresponding to Mob1 protein was hyperphosphorylated in cdc55Δ mutant cells. The Mob1 peptide contained two S/TP sites: S80 and T85 , suggesting that PP2ACdc55 is required to dephosphorylate Mob1 properly. Finally, we examined whether Cdc55 and Mob1 physically interact. Co-immunoprecipitation experiments showed that Mob1 co-purified with Cdc55 . However, Cdc5 protein levels were almost constant in the cdc55Δ mutant cells in anaphase. On the other hand, Clb2 was only partially degraded at 30 minutes in the cdc55Δ mutant cells, indicative of the first Clb2 degradation by APCCdc20. We conclude that APCCdh1 does not degrade Cdc5 and Clb2 efficiently in cdc55Δ mutant cells.To confirm this result, we also studied the activity of APCcdc55Δ mutant cells, but remained constant at later times since cdc55Δ mutant cells had a longer G1 phase . When the cells reached mitosis, the Bfa1-mCherry signal increased and the asymmetry became more evident. These results further confirmed the premature asymmetric Bfa1 localization in the absence of PP2ACdc55 function. Thus, PP2ACdc55 downregulation initiates MEN signalling by allowing Cdc5-dependent phosphorylation of Bfa1 and its asymmetric localization.Lastly, we studied the changes in Bfa1 localization during the cell cycle in the absence of PP2Aepletion . In wildhibition . cdc55Δ Cdc55 counteracts Bfa1 phosphorylation in metaphase, and upon downregulation of PP2ACdc55 in anaphase Bfa1 becomes hyperphosphorylated and located asymmetrically to the dSPB, the question arises as to why the exit from mitosis and MEN activation are not accelerated in cdc55Δ cells. It has recently been proposed that increased residence time of Tem1 on SPBs leads to premature Cdc15 loading but not to premature entry into anaphase cdc55Δ cells, we examined Cdc15 and Dbf2-Mob1 activation. Cdc15 kinase is dephosphorylated and activated by early released Cdc14 from the nucleolus. Cells were arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 ectopic expression (PP2ACdc55 inactivation). Cdc15 phosphorylation status was analyzed by western blot , the drug 1NM-PP1 was added to inhibit Cdk1. Remarkably, upon Cdk1 inactivation Mob1 was rapidly dephosphorylated . Therefore, a contribution of Zds1 to the PP2ARts1 complex is not expected.PP2ACdc55 counteracts Bfa1 Cdc5-dependent phosphorylation. Previous results have already shown that PP2ACdc55 can counteract Cdc5-dependent phosphorylation Cdc55 is not only a Cdk1–Clb2-counteracting phosphatase, but is also able to dephosphorylate Cdc5 targets.In the normal cell cycle, Bfa1 inactivation depends on Cdc5 phosphorylation. Although Bfa1 may be a substrate for other kinases that have not yet been described, our results indicate that PP2ACdc55. Mob1 is phosphorylated Cdc55 (our results). Progressive quantitative changes of the Cdc14/Cdk activity ratio during the course of mitotic exit cause dephosphorylation of individual substrates at distinct thresholds Cdc55 inactivation in which Cdc14 has already been released from the nucleolus Cdc55 inactivation does not entail premature MEN activation and mitotic exit. Cdc15 dephosphorylation patterns have often been used as a marker of MEN activity. However, we observed that Cdc15 is prematurely dephosphorylated and loaded onto the dSPB in cdc55Δ mutant cells and after Zds1-induced PP2ACdc55 inactivation. This is consistent with Cdc14 being released from the nucleolus prematurely during metaphase in cdc55Δ mutant cells. Despite Cdc15 being properly recruited to the dSPB, our results suggest that Cdc15 is not totally active as a kinase, since Dbf2-Mob1 is not recruited to the SPBs after PP2ACdc55 inactivation. In accordance with this, Cdc15 dephosphorylation never reaches the levels of wild-type cells in anaphase indicating that it is not fully active. In addition, Mob1 is not dephosphorylated upon PP2ACdc55 inactivation by Zds1 induction (despite Cdc14 being released), further confirming that Dbf2-Mob1 is not active in this condition. The study of late anaphase events confirmed the previous results. Cdh1, which is a late Cdc14 substrate, is not properly dephosphorylated, resulting in a defective APCCdh1 complex. Thus, APCCdh1 substrates such as Cdc5 Polo kinase and Clb2 are not efficiently degraded. These results led us to propose that there is an inhibitory input into the MEN cascade downstream of Bfa1 that acts as a break when Bfa1 is prematurely phosphorylated, thereby avoiding the untimely full activation of MEN. In fact, Cdk1-dependent Cdc15 inhibition has been postulated before cdc55Δ cells, as would be expected if MEN activity were inhibited by Cdk1–Clb2. Moreover, Mob1 was rapidly dephosphorylated upon Cdk1 inactivation after Zds1-dependent Cdc14 release. The above results led us to postulate that alleviation of the Bub2–Bfa1 inhibitory MEN signal is not enough to produce the premature exit from mitosis observed in cdc55Δ cells, and that the Cdk1–Clb2 inhibitory MEN signal is the additional mechanism that restrains MEN activity until anaphase. In this way, cells would initiate FEAR and MEN at anaphase onset upon PP2ACdc55 downregulation, but Cdk1–Clb2 would impose a break for MEN activation, and decreasing Cdk1 activity would be the mechanism that sequentially activates both pathways.Premature Bfa1 phosphorylation observed after PP2ACdh1 and Bub2–Bfa1 are both required to resequester Cdc14 into the nucleolus after mitotic exit Cdh1-dependent Cdc5 degradation is important to return Cdc14 to the nucleolus at the correct time, but it is not essential, since Cdc14 is resequestered into the nucleolus, albeit with a delay in cells lacking CDH1BUB2 and CDH1 eventually resequester Cdc14 into the nucleolus, suggesting that additional mechanisms regulate Cdc14 resequestration. PP2ACdc55 reactivation during late anaphase could be one of these additional mechanisms involved in the resequestration of Cdc14 into the nucleolus. We have demonstrated here that Cdh1 is not fully active since Cdc5 and Clb2 degradation are impaired in cdc55Δ mutant cells. Net1 dephosphorylation is required to reassociate to Cdc14 and resequester it into the nucleolus; Bfa1 dephosphorylation is needed to inactivate MEN and helps to return Cdc14 to the nucleolus. In cells lacking CDC55, Net1 is hyperphosphorylated for longer Cdc55-dependent dephosphorylation in late anaphase is important for properly resequestering Cdc14 into the nucleolus. Nevertheless, the cdc55Δ mutant cells exit from mitosis, despite their slower kinetics in resequestering Cdc14 to the nucleolus. Sic1 is accumulated at the correct time in the cells lacking CDC55 and its level remains constant for longer periods. Sic1 accumulation is probably sufficient to inhibit Cdk1 and to exit from mitosis in the absence of Cdc55. This is consistent with GAL-SIC1-db being sufficient to drive exit from mitosis in MEN mutants APCCdc55 promotes the first wave of Cdc14 release from the nucleolus and contributes to the accumulation of the Cdc5-phosphorylated form of Bfa1. In this way, FEAR promotes the activation of two important MEN components: (1) PP2ACdc55 downregulation facilitates Bfa1 inactivation, allowing Tem1 activation; and (2) the FEAR-induced Cdc14 released dephosphorylates Cdc15. Bfa1 asymmetric localization on the dSPB is induced upon Bfa1 phosphorylation and is important for the proper recruitment of MEN components to the dSPB. In addition, Tem1 binds to the mSPB and recruits Cdc15. Cdc15 itself recruits Cdk1 and Dbf2-Mob1 to the mSPB. In turn, Cdk1 phosphorylates Cdc15, which is dissociated from the mSPB and becomes asymmetrically located on the dSPB. At the same time, Cdk1 phosphorylates and inhibits Dbf2–Mob1. Our results indicate that Mob1 phosphorylation is also regulated by the phosphatase PP2ACdc55, helping to keep Dbf2–Mob1 inactive in early anaphase. In this way, Mob1 phosphorylation is constant despite the progressive decrease in Cdk1 activity because the counteracting phosphatase PP2ACdc55 is also downregulated. In late anaphase, the increase in Cdc14 activity and the decrease in Cdk1 activity alleviate the Cdk1 inhibitory signal towards Dbf2–Mob1, and the MEN is fully active. Upon its reactivation, PP2ACdc55 will also help keep Mob1 dephosphorylated.Our model, based on the current findings and those of previous published work, is summarized in Schizosaccharomyces pombe called the septation initiation network (SIN) (reviewed in S. pombe) and Sid2 (Mob1 orthologue) has recently been described Cdc55 in regulating the MEN and SIN pathways. Although the SIN is analogous to the MEN in budding yeast, its main role is to coordinate cytokinesis. However, in S. cerevisiae, the MEN also goes beyond mitotic exit and regulates cytokinesis. Several MEN components localized to the bud neck and were found to be required for the contraction of the actin-myosin ring Drosophila and vertebrates share elements of the Cdc15-like kinases  and Dbf2-Mob1-like kinases  and the Cdc14 protein phosphatase family . Some studies suggest that the Hippo pathway plays a role in regulating mitotic exit and cytokinesis, although the mechanism is not yet fully understood. Our studies of MEN regulation will contribute to our understanding of MEN-related pathways in other organisms. It will be interesting to discover whether MEN regulation by PP2ACdc55 is conserved among higher eukaryotes.Exit from mitosis is regulated by a conserved signalling pathway in CDC55 was N-terminal-tagged as previously described Y19FCDC28 mutant strains were created by integration and loop-out, via 5-FOA (5-fluoroorotic acid) selections. Zds1 in cells that had been arrested in metaphase by Cdc20 depletion was ectopically expressed as previously described All yeast strains used in this study were derivatives of W303. Epitope tagging of endogenous genes and gene deletions were performed by gene targeting using polymerase chain reaction (PCR) products. Endogenous For the Zds1-induction experiments cells were fixed in absolute ethanol throughout the experiment and rehydrated in minimum media before visualization. At least 50 cells were used to quantify the SPB ratios. For time-lapse microscopy, cells were incubated in minimum media throughout the experiment in an environmental chamber, and images were acquired every 5 minutes. A Zeiss Axio Observer Z1 inverted epifluorescence microscope with Apotome system equipped with an HXP 120C fluorescent lamp and a Carl Zeiss Plan-Apochromat 63× N.A 1.40 oil objective. Filters used were EGFP  and Cy3 . Z-stacks at 1-µm intervals were taken for each fluorescent channel and projected onto a single image per channel. SPB ratios were quantified as the signal intensity ratio between the two SPBs (dSPB relative intensity/mSPB relative intensity). SPB ratios less than or equal to 2 or greater than 2 were considered to represent symmetric or asymmetric localization, respectively. We used ZEN software for image acquisition and quantitative analysis of fluorescent microscopy .13C6-arginine and 100 mg/L 13C6-lysine (Cambridge Isotope Laboratories Inc.). Protein extracts were prepared by mechanical lysis using glass beads. Approximately 250 µg of the mixed heavy/light protein sample were processed for in-solution digestion as previously described Stable isotopes of yeast cells were labeled and protein extracts prepared as previously described 9 cells for Mob1 and 3.2×1010 cells for Bfa1. Protein extracts were prepared by mechanical lysis using glass beads. The clarified extracts were incubated with antibody, and the immunocomplexes were adsorbed onto magnetic protein-A Dynabeads® (Life Technologies). The beads were washed in extraction buffer and the protein-bound fraction eluted with SDS-PAGE loading buffer. The antibody used for immunoprecipitation was the α-Pk clone SV5-Pk1 (Serotec). For the phosphatase assays, Pk epitope-tagged Cdc14 was immunopurified on protein-A Dynabeads as above, and the beads were washed with phosphatase buffer . Reaction mix (phosphatase buffer containing 34 µM of DiFMUP) was added to the beads and incubated at 30°C for 2 min. Reactions were terminated by adding 200 mM sodium carbonate and the fluorescence of the samples was determined at 365 nm (absorption wavelength) and 455 nm (emission wavelength). Cdc14 recovered on the beads was quantified by western blot using an IRdye 800 coupled secondary antibody and the Odyssey Infrared Imaging System (Li-COR Biosciences). Phosphatase-specific activity was calculated as fluorescence units/protein levels and represented as arbitrary units.The immunoprecipitation assay was performed as described Protein extracts for western blots were obtained by NaOH or TCA protein extraction. Bfa1 and Cdc15 western blots where done using 10% protein gel at a 33.5∶0.3 acrylamide/bisacrylamide ratio. Antibodies used for western blots and immunofluorescence were the α-HA clone 12CA5 (Roche), α-HA clone 16B12 (Babco), Cdc14 (yE-17) sc-12045 (Santa Cruz Biotechnology), α-FLAG clone M2 (Sigma), α-Pk clone SV5-Pk1 (Serotec), Cdc5 (yC-19) sc-6733 (Santa Cruz Biotechnology), Sic1 (FL-284) sc-50441 (Santa Cruz Biotechnology), Clb2 (y-180) sc-907 (Santa Cruz Biotechnology), anti-tubulin clone YOL1/34 (Serotec) and anti-phosphoglycerate kinase (Life Technologies). The secondary antibodies were Cy3-labeled α-mouse , fluorescein-conjugated α-rat (Millipore) and Cy3-labeled α-goat (Jackson ImmunoResearch).Figure S1Cdc55 promotes Bfa1 phosphorylation. Strain Y532 (3-ESP1 CDC14-Pk9 BFA1-HA6MATa MET-CDC20 GAL1-Flag) was arrested in metaphase by Cdc20 depletion, and separase ectopic expression was induced by galactose addition. Bfa1 phosphorylation and Esp1 expression levels were analyzed by western blot. Cdc14 release was monitored by immunofluorescence as control of PP2ACdc55 inactivation.Separase-dependent inactivation of PP2A(TIF)Click here for additional data file.Figure S2Cdc55 inactivation. Strains Y1034 (3-ZDS1 CDC14-Pk9 BFA1-HA6MATa MET-CDC20 GAL1-Flag) and Y1033  were arrested in metaphase by Cdc20 depletion and shifted to 37°C for 180 min before Zds1 induction. Bfa1 phosphorylation and Zds1 expression levels were analyzed by western blot. Cdc14 release was monitored by immunofluorescence.Cdc5 Polo-like kinase-dependent Bfa1 phosphorylation is required for Zds1-induced PP2A(TIF)Click here for additional data file.Figure S3Cdc55 inactivation does not depend on the kinase Kin4. Strains Y597 (3-ZDS1 CDC14-Pk9 BFA1-HA6MATa MET-CDC20 GAL1-Flag) and Y563 (as Y597 but kin4Δ) were arrested in metaphase by Cdc20 depletion and Zds1 ectopic expression was induced by galactose addition. Bfa1 phosphorylation and Zds1 expression levels were analyzed by western blot. Cdc14 release was monitored by immunofluorescence as control of PP2ACdc55 inactivation.Bfa1 phosphorylation observed upon PP2A(TIF)Click here for additional data file.Figure S4Zds1 protein levels. Zds1-induction controls of experiments in (TIF)Click here for additional data file.Figure S59 CDC15-eGFP BFA1-mCherryMATa CDC28F19 CDC14-myc) and Y957  were arrested at G1 with α-factor and released into a synchronous cell cycle. Time-lapse microscopy was performed as described in the Material and Methods.Increased Cdc15 asymmetric localization in the absence of Cdc55 in a synchronous cell cycle after G1 release. Strains Y911 ((TIF)Click here for additional data file.


Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of eukaryotic initiation factor-2α (eIF2α). Evidence is accumulating that persistent eIF2α phosphorylation at Ser51 through prolonged overactivation of regulatory kinases occurs in neurodegenerative diseases such as Alzheimer’s disease (AD), leading to shutdown of general translation and translational activation of a subset of mRNAs. Recent advances in the development of gene-based strategies and bioavailable inhibitors, which specifically target one of the eIF2α kinases, have enabled us to investigate pathogenic roles of dysregulated eIF2α phosphorylation pathways. This review provides an overview of animal model studies in this field, focusing particularly on molecular mechanisms by which the dysregulation of eIF2α kinases may account for synaptic and memory deficits associated with AD. A growing body of evidence suggests that correcting aberrant eIF2α kinase activities may serve as disease-modifying therapeutic interventions to treat AD and related cognitive disorders. Although the molecular cause of Alzheimer’s disease (AD) has not been completely understood, recent investigations increasingly implicate the aberrant translational machinery through the α subunit of eukaryotic initiation factor-2 (eIF2α) in the pathogenesis of this devastating neurodegenerative disease. It has been reported that eIF2α phosphorylation is significantly increased in the brains of sporadic AD patients as well as different lines of amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mice . AccumulWhile eIF2α phosphorylation suppresses general protein synthesis, it is shown to paradoxically cause translational activation of a subset of mRNAs, including the β-secretase called β-site APP-cleaving enzyme 1 BACE1;  and the iMet (initiator methionyl-tRNA) to form a ternary complex, which delivers the initiator tRNA to the 40S ribosomal subunit. Exchange of GDP for GTP on the γ subunit is catalyzed by eIF2B, a guanine nucleotide exchange factor that is required to replenish the active GTP-bound form of eIF2 complex for a new round of translational initiation. Phosphorylation of eIF2 on its α subunit at Ser51 in response to diverse stress stimuli suppresses general translation initiation, since it converts eIF2 to a competitive inhibitor of eIF2B by blocking the GDP-GTP exchange reaction and reducing the dissociation rate of eIF2 from eIF2B . Although phosphorylation of eIF2α causes a reduction in general translation, it also selectively increases the translation of a subset of mRNAs that contain upstream open reading frames (uORFs). The molecules that undergo the gene-specific translational upregulation via eIF2α phosphorylation include the β-secretase enzyme BACE1 . The molecular mechanism of eukaryotic translation initiation and its regulation have been described in detail in previous reviews  and binds GTP and Met-tRNAme BACE1  and the me BACE1 , which a reviews .Figure 1; PERK is primarily activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), a phenomenon termed ER stress. Generally, PERK-dependent eIF2α phosphorylation is thought to block global translation initiation and in this manner can alleviate ER stress by reducing the amount of protein transport into the ER. This allows the ER time to refold misfolded proteins and dispose of those that are terminally misfolded, an important element of cellular protective UPR processes. PKR was initially discovered as a kinase that phosphorylates eIF2α in response to viral infection or double-stranded RNA, thereby blocking the translation of viral mRNAs and promoting apoptosis. GCN2 is primarily a sensor of amino acid availability and a regulator of changes in gene expression in response to amino acid deprivation. GCN2 is also activated by UV irradiation and viral infection. HRI is predominantly found in erythroid cells and activated by heme deficiency. Remarkably, considerable evidence is accumulating that these eIF2α kinases are dysregulated on different disease conditions and may play pathogenic roles, especially, in neurodegenerative disorders including AD and related cognitive impairments, as summarized in this review.The phosphorylation of eIF2α at Ser51 is controlled by four protein kinases such as general control non-derepressible-2 kinase (GCN2), double-stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase  show elevations in eIF2α phosphorylation in brains and deficient learning and memory, as compared with age-matched ApoE3 control mice . This Ap+/S51A point mutation also enhances L-LTP and memory formation in multiple learning paradigms such as the Morris water maze, fear conditioning and conditioned taste aversion , which represent an early onset and aggressive amyloid mouse model based on a combination of five familial AD (FAD) mutations , 2007. O-/- and GCN2+/- deficiencies aggravate rather than suppress eIF2α phosphorylation in 5XFAD mice, thus failing to rescue memory deficits in the contextual fear conditioning task (-/- mice show reduced eIF2α phosphorylation (relative to wild-type controls) without compensatory changes in phosphorylated PERK levels. Therefore, we postulate that signaling mechanisms controlling eIF2α phosphorylation are different between normal and severe AD conditions. GCN2 may be an important eIF2α kinase under the physiological condition, whereas PERK-mediated eIF2α phosphorylation becomes prominent under exposure to great amounts of misfolded proteins  and GCN2 may function as a negative regulator of this pathway  in APP/PS1 mice . The resin silico model reveals that chronic and severe ER stress induces persistent PERK-dependent phosphorylation of eIF2α, which is sufficient to induce shutdown of translation . APP/PS1 mice with conditional PERK ablation also have reduced levels of Aβ and C99 (Arctium lappa (L.), to APP/PS1 mice can block the translational upregulation of BACE1 by suppressing the PERK-eIF2α pathway in the absence of transcriptional alteration (in vitro model of incipient AD conditions (Which eIF2α kinase(s) may be responsible for mediating BACE1 upregulation? We recently demonstrate that reducing PERK-dependent eIF2α phosphorylation blocks BACE1 elevation in advanced stages of 5XFAD mice (8-to 9-month-old), leading to the suppression of β-amyloidogenesis as evidenced by decreased levels of the β-cleaved C-terminal fragment of APP (β-CTF or C99), Aβ40 and Aβ42 peptides, and amyloid plaque burden in PERK and C99 . Furtherteration . This acteration . Furtherteration , 2012. Inditions . Therefoin vivo and cell culture experiments reveal that infection with herpes simplex virus type 1 (HSV1: a virus suggested to be implicated in AD development) causes activation of the PKR-eIF2α pathway, resulting in elevations of BACE1 expression and Aβ/C99 generation  surrounding plaques, which in turn further accelerates Aβ generation in 5XFAD mouse and human AD brains . TherefoATF4, a repressor of CREB (CREB-2), is another signaling molecule of which translation is facilitated by eIF2α phosphorylation . As descFigure 1). It should be noted that cross-talk impacts such as BACE1- or Aβ-dependent suppression of CREB function (The findings summarized in this review provide experimental evidence that the aberrant activation of eIF2α phosphorylation pathways found in AD may be responsible for multifaceted memory-deteriorating and neurodegenerative mechanisms, including accelerated β-amyloidogenesis through BACE1 elevation, CREB dysfunction via ATF4 upregulation, and inhibition of general translation (function  would wofunction  and PKR function , 2012 arThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We developed an automated method using depth and one axis of body acceleration data recorded by animal-borne data loggers to identify activities of penguins over long-term deployments. Using this technique, we evaluated the activity time budget of emperor penguins (n = 10) both in water and on sea ice during foraging trips in chick-rearing season. During the foraging trips, emperor penguins alternated dive bouts (4.8±4.5 h) and rest periods on sea ice (2.5±2.3 h). After recorder deployment and release near the colony, the birds spent 17.9±8.4% of their time traveling until they reached the ice edge. Once at the ice edge, they stayed there more than 4 hours before the first dive. After the first dive, the mean proportions of time spent on the ice and in water were 30.8±7.4% and 69.2±7.4%, respectively. When in the water, they spent 67.9±3.1% of time making dives deeper than 5 m. Dive activity had no typical diurnal pattern for individual birds. While in the water between dives, the birds had short resting periods (1.2±1.7 min) and periods of swimming at depths shallower than 5 m (0.25±0.38 min). When the birds were on the ice, they primarily used time for resting (90.3±4.1% of time) and spent only 9.7±4.1% of time traveling. Thus, it appears that, during foraging trips at sea, emperor penguins traveled during dives >5 m depth, and that sea ice was primarily used for resting. Sea ice probably provides refuge from natural predators such as leopard seals. We also suggest that 24 hours of sunlight and the cycling of dive bouts with short rest periods on sea ice allow emperor penguins to dive continuously throughout the day during foraging trips to sea. Pygoscelis papua) have increased in number and expanded their range southward, while the ice-dependent species such as Adélie penguins (Pygoscelis adeliae) has decreased at almost all locations on the Antarctic Peninsula and contracted their distributional range poleward Penguins are widely distributed around the Southern Ocean, and they are important consumers in the marine ecosystem Aptenodytes forsteri) is considered an ice-dependent species, because it uses fast ice as breeding ground. For specific responses of emperor penguins to sea ice condition, the population dynamics and the breeding success have been studied The emperor penguin . The birds had a mean mass of 24.6±2.4 kg, all of which were increased by 2.9±1.3 kg at the retrieval , which showed that resting time on the ice increased with duration of dive bouts. On the other hand, for the models with traveling time on the ice as dependent variable, difference of AICc between models with any fixed effect and random effect only was lower than 2.0. In addition, the wi of each model was lower than 0.4. Thus, resting time on the ice was best explained by the time duration of the previous dive bout. On the other hand, traveling time on the ice between dive bouts was not clearly related to one single factor: the duration of the previous dive bout, the cumulative diving time, and the cumulative bottom time each gave similar model fits.Results of LMMs to investigate relationships between activities on the ice and those during previous dive bouts are shown in This is the first report for the activity time budget both in water and on sea ice of breeding emperor penguins on foraging trips during chick-rearing period. Our results showed that emperor penguins made repeated dive bouts and rest periods on sea ice over a prolonged period. Although it is known that emperor penguins perform repeated dive bouts during foraging trips at sea Hydrurga leptonyx) are high at sea ice edges Our results show that the penguins primarily rested on sea ice. Continuous travel over long distances did not occur on sea ice. We also found that after leaving the colony and arriving at the ice edge prior to the start of a foraging trip, birds spent 3 to 38 hours waiting before making the first dive. We suspect that rest on the sea ice and prolonged waits at the ice edge are secondary to the presence of predators. For Antarctic penguins, predation risks by leopard seals  during the chick-rearing period from 26 October to 24 November 2005. Fourteen birds departing the colony were captured near the edge of the colony. Data loggers were attached to the central back feathers using waterproof Tesa™ tape  and stainless steel cable ties. Eleven birds were equipped with acceleration data loggers  which recorded depth (1 Hz), temperature (1 Hz), speed (1 Hz), and two axes of acceleration . Three birds were equipped with 3-D loggers  which recorded depth (1 Hz), temperature (1 Hz), three axes acceleration and three axes geomagnetism . The devices were set to start recording 4–96 h after deployment by the preset delay timer. According to the limitation of memory size, the logger cannot cover the whole foraging trip of each individual. The variation of recording start time (4–96 h) enabled us to record all phases of the foraging trips. VHF transmitters  were attached to the lower back with cable ties. Body mass was measured to the nearest 100 g at deployment and recapture, using a balance (Pesola 50 kg). Mass of the devices deployed were less than 1% of the body mass. Methods of the field study and the deployments of loggers were described in detail in Penguins’ activities were categorized every 4 seconds using the depth and longitudinal axis of acceleration . We put Loggers measured both specific acceleration  and gravity-related acceleration Data retrieved from the loggers were analyzed using a customized macro program in IGOR PRO version 6 . We also used Ethographer In the procedures, it was first determined whether a bird was on ice or at the water surface (depth <0.5 m). Then, the maximum pitch angle during a period between the first and end points at which depths were less than the threshold (depth = 0.5 m) was calculated. A pitch angle of ≥50° was used as a criterion for walking or standing because the distribution of pitch angle was bimodal with higher values (≥50°) corresponding to walking and standing on ice . If the When a bird was on the ice, activities were categorized into four types according to the pitch angle and traveling activity derived by the k-means cluster analysis . When a When the bird was diving (the depth ≥5 m), activities were categorized into three types: descent, bottom, and ascent phases, according to the time series analysis of depth data. Bottom time, which was assumed to be the time spent in the foraging area, was defined as the duration from the first ascent to last descent in each dive −1−1Statistical analysis was performed based on dive bouts. If a bird exited the water and was on ice, the next dive was placed into a new dive bout. During the time between deployment/release near the colony and arrival at the ice edge, the proportion of time spent in each activity was calculated. When the bird was on the ice, traveling distance was estimated with accumulative time and mean walking speed 0.5 ms−1 and tobowi) for each model, which represents the relative likelihood of candidate models wiTo investigate relationships between activities on the ice and the previous dive bout, we used a linear mixed model (LMM) fit by maximum likelihood with a Gaussian error distribution, identity link function, and bird ID as random effect for the intercept. When a bird stayed on the ice until the next dive bout, accumulative time for resting: standing and lying on the ice, and accumulative time for traveling: walking and tobogganing, were calculated and respectively compared with time duration, accumulated time of diving, and bottom time duration during the previous dive bout. Models were ranked based on their Akaike Information Criteria corrected for small sample sizes (AICc) and we calculated the Akaike weight (Movie S1It shows a group of emperor penguins synchronously diving from ice edge.(MOV)Click here for additional data file.Movie S2It shows a leopard seal looking for emperor penguins at the edge of the water.(MOV)Click here for additional data file.


The brain-enriched microtubule-associated protein tau, a critical regulator of cytoskeletal dynamics, forms insoluble aggregates in a number of neurodegenerative diseases termed tauopathies, including Alzheimer's disease (AD). Hyperphosphorylation of tau protein is an important mechanism for aggregation, so many studies on the pathogenesis of AD and other tauopathies have focused on regulation of tau phosphorylation by kinases and phosphatases. Less studied are mechanisms of tau transcriptional and post-transcriptional regulation by extracellular signals such as BDNF and how such changes alter neuronal function. Previously, we reported that tau is required for morphological plasticity induced by BDNF. Here, we further explore tau modification during BDNF-induced changes in neuronal cell morphology. In undifferentiated SH-SY5Y cells lacking neurites, tau formed a sphere within the soma as revealed by immunocytochemistry. In contrast, tau was enriched in the neurites and sparse in the soma of SH-SY5Y cells induced to differentiate by retinoic acid (RA). Treatment with RA also increased total tau protein levels but decreased expression of tau phosphorylated at Ser262 as determined by Western blot. Both effects were further enhanced by subsequent BDNF treatment. Upregulation of tau protein and downregulation of p-Ser262 tau were correlated with total neurite length . When primary E18 hippocampal neurons were treated with nocodazole, a blocker of microtubule polymerization, nascent neurites were lost and tau shifted to the soma. This process of retrograde tau movement away from neurites was reversed by BDNF. These results indicate that tau is redistributed to neurites and dephosphorylated during RA- and BDNF-mediated differentiation. Dysregulation of the protein tau is implicated in the pathogenesis Alzheimer's disease (AD) and many other neurodegenerative disease Tau is essential for the maintenance of polarity and for the survival of mature neurons Some familial neurodegenerative diseases have been linked to tau gene (MAPT) mutations that lead to alternative splicing, changes in phosphorylation state, reduced affinity for tubulin, and (or) enhanced capacity for self-association into filaments and aggregates In addition to MT stabilization, tau protein interacts with many signaling pathways to regulate the intracellular trafficking of organelles and molecule involved in presynaptic and postsynaptic function 2 humidified atmosphere at 37°C. Cells were grown with or without all tans-retinoid acid (RA) for 5 days before treatment.Human SH-SY5Y neuroblastoma cells from ATCC were cultured in Dulbecco's modified Eagle's medium / F12 medium  with 10% heat-inactivated fetal bovine serum , 1% v / v penicillin / streptomycin  in a 5% CO2 until use. Neurobasal and B27 supplement were purchased from Invitrogen .Primary hippocampal neurons were prepared from E18 rat fetuses as previously described All experiments involving the use of animals were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and animal care guidelines approved by the Institute of Experimental Medicine ASCR Animal Care Committee. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Third Military Medical University. All efforts were made to minimize animal suffering and to reduce the number of animals used.BDNF, RA, taxol and nocodazole were purchased from Sigma–Aldrich . BDNF was dissolved in sterilized water at 200 nM. Taxol and nocodazole were dissolved in DMSO  at 1 mM and 20 mM separately. Taxol and nocodazole solution were stored at 4°C and BDNF solution was stored at −20°C until use.Primary cultured hippocampal neurons were pre-incubated for 2 h with taxol or nocodazole at the following concentration before BDNF incubation: taxol (10 nM), nocodazole (100 nM for 3 day in vitro (DIV) neurons, 500 nM for 13DIV neurons.Western blot was performed as previously described z-axis. Images were processed using Adobe Photoshop 3CS . Immunofluorecence intensity measurement was performed with the aid of LAS-AF software . Immunofluorescence of tau and α-tubulin in the soma part of each neuron was analyzed. The ratio of average intensity of tau and tubulin was recorded. Data were shown as mean ratio ± SD (n = 40).Immunocytochemistry was performed as previously described Neurite length of hippocampal neurons was measured as previously described All the quantitative data were obtained from three independent experiments and presented as the mean ± SD. Statistical significances of differences between two groups were determined by using unpaired Student's t test. All P values were two sided, and P<0.05 was considered as statistical significance.Changes in the subcellular distribution and conformation of tau during the progression of tauopathies have been studied extensively in an attempt to understand the process of neurofibrillary tangle (NFT) formation, the aberrant conformation of tau that is a hallmark of these diseases These immunostaining studies in differentiating SH-SY5Y cells suggests that tau protein expression is upregulated and shifted away from the soma during neurite formation, possibly due to reduced phosphorylation and enhanced association with α-tubulin. Tau protein phosphorylation at Ser262 greatly influences the binding of tau to MTs 2 = 0.9622, 2 = 0.8796, Tau binding to MTs is a critical regulator of the balance between MT polymerization and depolymerization, and thus a major influence on cytoskeletal structure and cell morphology Increased phosphorylation and redistribution of tau may be a common response to neuronal stress The redistribution of tau protein was then examined in neurons with different degrees of nocodazole damage as indicated by the extent of neurite retraction. Compared to neurons with long dendrites , tau immCollectively, these results suggest that subcellular tau distribution is correlated with Ser262 phosphorylation status, dendritic length, and the balance between MT polymerization and depolymerization. Conditions that favor MT depolymerization and tau Ser262 phosphorylation are associated with accumulation of tau in spherical soma inclusions. Retinoic acid and BDNF appears to at least partially reverse these changes, suggesting a novel mechanism for the neuroprotective efficacy of these agents.Brain-derived neurotrophic factor is neuroprotective against a variety of insults. In this study, we report that BDNF can increase tau protein expression in RA-differentiated SH-SY5Y cells ,3 as welThe spherical tau aggregates we observed in undifferentiated SH-SY5Y cells  may sharOur data also highlight BDNF signaling as a feasible target for drugs against tauopathies The BDNF signaling cascade, including BDNF, TrkB, and downstream kinases and phosphatases are targets for drugs modifying tau expression, phosphorylation, and cellular distribution. Indeed, such treatments could benefit a broad spectrum of tauopathies, including AD We conclude that tau protein is a critical mediator of BDNF-induced morphological transformation and possibly also of BDNF-mediated neuroprotection. Since BDNF influences neuronal activity, function, and survival throughout life, much further study is warranted to examine BDNF-tau interactions in sporadic, genetic, and age-related neurodegenerative diseases. Development of transgenic animal models would greatly facilitate such studies.


High concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE.For In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn −/− mice (Wsh Rec Fyn −/−).de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn −/− BMMCs.Monomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and  High concentrations of plasmatic IgE are related to a number of systemic inflammatory conditions  and receMast cells (MCs) are central players in allergic reactions and also constitute a well documented component of tumor microenvironment ,5. The pThe best characterized stimulus for MC activation is the crosslinking of the high affinity IgE receptor (FcϵRI), which occurs after the interaction of IgE bound to the receptor with specific antigens . After Fin vitro studies have shown, however, that non-specific IgE, in the absence of antigen, is able to modify MC secretory profile in distinct cell preparations and cell lines. Those changes, produced by IgEs with no relevant recognition for specific antigens, have been hypothesized to be relevant to the initiation of local inflammatory reactions, especially in humans with high levels of circulating IgE, like atopic individuals [The initial binding of monomeric IgE to the receptor was considered for long time as a “sensitization” event for late antigen-dependent stimulation. Recent ividuals ,11. HoweMC activation is closely related to tumor growth ,13. Specin vitro; 2) to analyze if monomeric IgE could exacerbate the pro-tumorigenic properties of this cell type in vivo; and 3) to investigate some of the molecular mechanisms underlying the effects of IgE on VEGF production and tumor growth.The objective of this work was threefold 1) to test if monomeric IgE (in the absence of antigen) could induce the production of VEGF in MC de novo transcription and transport from endoplasmic reticulum to the Golgi apparatus [Production of angiogenic factors by MC has been shown to occur few hours after different stimuli, such as hypoxia, antigen or PMA ,17. To ipparatus  was needpparatus , and PP2Two main Src family kinases modulate mediator secretion from MCs. Lyn and Fyn kinases have been shown to be involved in early signaling after FcϵRI crosslinking, leading to the activation of downstream pathways regulating pro-inflammatory cytokine production . In ordeIn order to investigate the role of Fyn kinase in the IgE-dependent VEGF synthesis, BMMCs derived from WT or Fyn −/− mice were incubated at 37°C in the presence of vehicle or IgE (1000 ng/ml) for 8 hours. Cells were collected using a cytospin centrifuge and processed to detect VEGF by fluorescent immunostaining. Figures 164 mRNA, the predominant isoform in different cell systems including MC [In order to investigate if the absence of Fyn could lead to alterations on IgE-dependent VEGF mRNA synthesis, we analyzed VEGF mRNA content by RT-PCR in WT and Fyn −/− BMMCs after IgE stimulation. Optimal conditions for amplification of VEGF and GAPDH (control) were experimentally determined in our cell system (see Methods section). VEGFuding MC , was incTo test the hypothesis that IgE-induced VEGF protein translation could be defective in the absence of Fyn, we evaluated whether VEGF synthesis in Fyn −/− cells could be more sensitive to the protein synthesis inhibitor cycloheximide (CHX) than in WT BMMCs. We found that CXH pre-treatment provoked a decrease on VEGF production in both cell types, but Fyn−/− BMMCs showed an increased sensitivity to this inhibitor Figure A. The maIt has been shown that VEGF synthesis under different stressful conditions (such as hypoxia) depends on the rapid modification of protein translation machinery . In thosFigure in vivo, we took advantage of the well-recognized role of MC and VEGF on the development of B16 melanoma tumor [In order to evaluate the effect of IgE on the secretion of VEGF in MC and the role of Fyn kinase in that process ma tumor ,23. A numa tumor . For insma tumor .We started defining the amount of IgE that could be administered in mice to analyze its effect on melanoma tumor growth. We decided to utilize a dose of IgE that could give a measurable parameter of MC and basophil activation. Passive anaphylactic reactions have been shown to depend on MC activity when initiated with an specific IgE and triggered with the corresponding antigen ,26. ExogWe determined the concentration of IgE needed for maximal activation of passive anaphylactic reaction in C57BL/6J mice. Monomeric IgE directed against DNP-HSA was intravenously (i.v.) injected to mice and twenty four hours later, antigen was i.v. injected in the presence of Evans blue dye. Twenty minutes after antigen administration, limbs were removed and dye extravasation was measured. As can be observed in Figure To evaluate the effect of IgE on melanoma tumor growth, C57BL6/J (WT) mice were treated with a single i.v. administration of saline or monomeric IgE (750 ng/mouse) and twenty four hours later, mice were subcutaneously (s.c.) inoculated with B16 melanoma cells in one ear pinna and generated tumors were removed after four weeks. Tumors obtained from IgE-treated mice (+IgE) were larger than those obtained from saline-treated mice (−IgE) , the size of the tumors after 28 days was significantly lower than the observed in WT mice . IgE-dependent tumor growth was significantly impaired in mice reconstituted with Fyn−/− BMMCs compared to mice reconstituted with WT cells Figure A-B. A deA possible link between inflammation and cancer was first proposed in the nineteenth century, based on observations that tumors often arose at sites of chronic inflammation and that inflammatory cells were present in biopsies from tumors . Since tin vivo.In this paper we show that monomeric IgE (with irrelevant capacity of tumor recognition and in the absence of antigen) induces VEGF production in isolated MC through a Fyn kinase-dependent mechanism, and this exacerbates pro-tumorigenic properties of this particular cell type . Although high concentrations of monomeric IgE have been shown to activate MC leading to the production of inflammatory mediators [Our results show, for the first time, that nonspecific monomeric IgE promotes the synthesis of pro-angiogenic factors modulating the protein translation system in MCediators -31 we wePharmacological characterization of IgE-induced VEGF production indicates that this factor is transported from ER-to-Golgi and is secreted in vesicles decorated with TTx-sensitive VAMPs. This mechanism resembles the reported pathway of hypoxia-induced VEGF secretion in BMMCs . The obsThe modifications of the translational apparatus associated to VEGF production were analyzed and it was found that monomeric IgE induced dephosphorylation of 4E-BP1 in WT MC, suggesting an increase on IRES-dependent protein translation. In contrast, 4E-BP1 dephosphorylation was not observable in the absence of Fyn, where even an increase on 4E-BP1 phosphorylation was detected after IgE treatment. Our data show for the first time that monomeric IgE causes changes on the translational machinery of MC, favoring IRES-dependent protein translation, in a Fyn dependent-manner. Since the IRES-dependent mechanism of protein translation occurs preferentially during hypoxic conditions , it is pin vivo, an increased size of melanoma tumors was observed in WT and in Wsh-reconstituted mice. To our knowledge, this is the first report showing that non-specific IgE promotes melanoma tumor growth and angiogenesis in a MC-dependent fashion. Loading MC with circulating IgE has been proposed to rapidly occur due to the formation of specific cellular structures in MC that are able to penetrate the endothelial cell layer of blood vessels [When IgE was administered  vessels . On the  vessels ,30, so, MC-derived VEGF seems to contribute to active angiogenic processes observed in some tumors . The effWv/Wv) [The positive influence of MC on tumor growth has been demonstrated utilizing MC-deficient mice (KitWv/Wv) . Our resWv/Wv) , confirmFyn kinase is an important effector of FcϵRI signaling system. Its activation after IgE/Antigen stimulation of MC leads to degranulation, leukotriene synthesis and selective cytokine expression ,35. Howein vitro[Administration of tumor-specific mouse monoclonal IgE antibodies prevent the development of mammary adenocarcinoma  and inhiin vitro. Since iin vitro, our datin vitro.Our data suggest that monomeric IgE is able to potentiate the pro-tumorigenic properties of MC in a Fyn kinase-dependent fashion Figure . TherefoSalts, inhibitors and buffer components, as well as anti-dinitrophenyl (DNP) IgE (clone SPE-7) and DNP coupled to human serum albumin (DNP-HSA) were purchased from Sigma-Aldrich. Recombinant IL-3 was purchased from Peprotech.KitW-sh , 129/Sv-Lyntm1Sor/J (stock No. 003204) and 129-Fyntm1Sor/J mice (stock No. 002271) were purchased from Jackson Laboratories. 129-Fyntm1Sor/J mice were back-crossed with C57BL/6J at least five times in our animal facilities to obtain Fyn-deficient animals (Fyn −/−) on the C57BL/6J genetic background. Genotyping of each animal was performed by PCR from genomic DNA utilizing the primers and conditions suggested by the provider. Animals were maintained under pathogen-free conditions and treated in accordance with NIH guidelines. Experiments were performed with age-matched male mice of at least 8 weeks old and were approved by the animal ethics committee of Cinvestav . When necessary, mice were euthanized by CO2 inhalation.C57BL/6J , mast cell-deficient B.6Cg-2, 1 mM MgCl2, 5.6 mM glucose, 0.05% bovine serum albumin; BSA) at 37°C. After incubation, different concentrations of the specific antigen  were added. After 30 minutes at 37°C, cell supernatants were collected and tested for β-hexosaminidase activity as described [Bone marrow-derived mast cells (BMMCs) were generated extracting the bone marrow from both tibias of mice four to eight weeks old. Bone marrow was cultured in BMMC media  during four weeks and after that, FcϵRI receptor expression was analyzed by flow cytometry utilizing a specific positive for the IgE receptor as described . To confescribed .Two million BMMCs were incubated with distinct concentrations of IgE for different times at 37°C in 1 ml of BMMC media supplemented with a protease inhibitor cocktail . VEGF was determined in the supernatants utilizing ELISA kits from Peprotech and Invitrogen. Actinomicyn D , Brefeldin A , Tetanus toxin  and PP2 (10 μM) were added to the cells 15 min previous to the stimulation  with 100Two million BMMCs were incubated with 1000 ng/ml IgE at 37°C for different times. Total RNA was isolated utilizing Tri-Reagent and isopropanol RNA precipitation as described . cDNA syTwo million BMMCs were incubated with 1000 ng/ml IgE for 8 h in 2 ml of BMMC media supplemented with a protease inhibitor cocktail (Roche) at 37°C. Cells were collected by centrifugation using a cytospin, then washed, fixed in cold acetone for 5 min at 4°C, washed again and blocked with 3% BSA, 2% goat serum and 0.01% Tween-20 in PBS for 30 min at RT. Cells were incubated with an anti-VEGF rabbit polyclonal serum  diluted in blocking solution overnight at 4°C. After washes with PBS, slides were treated with an Alexa 568-anti-goat secondary antibody  diluted in blocking solution for one hour. Finally, an incubation with 4′, 6-diamidino-2-phenylindole, dihydrochloride  for 5 min at RT was performed and slides were properly mounted. A confocal microscope (Olympus FluoView FV1000) with a 405-nm laser for DAPI and 543–nm laser for Alexa 568 was used. Fluorophores were imaged using a sequential line scan, with detection bands set at 405 to 461 nm for DAPI stain and 543 to 603 nm for Alexa 568. Each image was saved at a resolution of 512 × 512 pixel image size and analyzed. Average fluorescence intensities of VEGF per cell were measured from 13 to 33 cells per experimental group utilizing the software LabWorks 4.5.Two million BMMCs were incubated with 1000 ng/ml IgE for 0, 15, 30 and 60 min in Tyrode’s buffer without BSA  at 37° . Cell pein vivo. C57BL/6J mice were i.v. injected with saline solution or, 25, 50, 250, 500, 750 and 1000 ng of anti-DNP IgE dissolved in 100 μL of sterile saline solution. Then, a passive anaphylaxis assay [In order to evaluate the role of IgE on melanoma tumor growth, we first determined the amount of IgE that could be needed to occupy the free FcϵRI receptor on the surface of mast cells is assay  was perfis assay . To test6 cells) in Tyrode’s buffer without BSA and four weeks after adoptive transfer of BMMCs MC reconstitution was confirmed by histological analysis of ear pinna and passive cutaneous anaphylactic reactions. Wsh mice were reconstituted with BMMCs obtained from C57BL/6J (Wsh Rec WT) or C57BL/6J-Fyn−/− (Wsh Rec Fyn−/−) mice. Animals were inoculated with melanoma cells four to six weeks after reconstitution and tumor growth (see following section) was compared with age-matched WT and non-reconstituted animals.MC-deficient (Wsh) mice (eight to twelve weeks old) were reconstituted with MC as described . Briefly6 B16 melanoma cells in Tyrode’s buffer without BSA and the right ear pinna with vehicle [B16-F1 melanoma cells obtained from ATCC  were generously donated by Dr. Guadalupe Reyes, Cinvestav, and cultured as suggested by the provider. Briefly, cells were propagated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 100 U of penicillin and 100 μg/ml of streptomycin [ vehicle . To test vehicle ).i.e. vessels containing only the endothelial cell layer were considered small vessels, whereas those with the smooth muscle cell layer were considered to be large blood vessels [Biopsy specimens of the ears inoculated with vehicle or B16 melanoma cells were fixed in neutral buffered formalin  prior to paraffin embedding. Sections of 2.5 μm were cut and examined. For each treated animal (n = 2-7 per group), some ear sections were stained with hematoxylin and eosin (H&E) and other sections were stained with 0.1% toluidine blue (TB), pH 1.0  in order vessels .Unless specified otherwise, all data are expressed as the mean ± SEM of at least three independent experiments. Student’s t-test or Two-Way ANOVA followed by Student-Newman Keuls test was used to evaluate the significance of the differences. Statistically significant was set at P < 0.05.MC: Mast cells; IgE: Immunoglobulin E; FcϵRI: High affinity IgE receptor; PMA: Phorbol myristate acetate; PI3K: Phosphatidylinositol-3-kinase; TNF: Tumor necrosis factor; IL-3: Interleukin-3; CCL2: Chemokine (C-C motif) ligand 2; BMMCs: Bone marrow-derived mast cells; VEGF: Vascular endothelial growth factor; S1P: Sphingosine 1 phosphate; DNP: Anti-dinitrophenol; DNP-HSA: DNP coupled to human serum albumin; Act D: Actinomicyn D; BFA: Brefeldin A; TTx: Tetanus Toxin; DAPI: 4′, 6-diamidino-2-phenylindole, dihydrochloride; S6: Ribosomal protein S6; 4E-BP1: Eukaryotic initiation factor 4E binding protein 1; WT: Wild type; Fyn −/−: Fyn-deficient; Wsh: Mast cell-deficient B.6Cg-KitW-sh mice; i.v.: Intravenously; s.c: Subcutaneously; DMEM: Dulbecco’s modified Eagle medium.The authors declare that they have no competing interests.in vitro studies, contributed to the interpretation of the results, participated in the statistical analysis of the study and drafted the manuscript. AI-S generated BMMCs, carried out passive cutaneous anaphylaxis studies and Western blots, determined VEGF mRNA quantification and VEGF secretion. DG performed blood vessels quantification. ML contributed to the design of the experiments, provided vital subjects for research and edited manuscript. CG-E conceived the study, designed the research, coordinate the activities of each contributor, provided vital subjects for research and edited manuscript. All authors read and approved the final manuscript.GYJ-A contributed to the design of the experiments, carried out immunofluorescence, performed the tumor studies, participated in the


Additionally, cystatin C-based GFR was higher than that calculated from creatinine level (p < 0.0001) in HNF1A-MODY, while the two GFR estimates were similar or cystatin C-based lower in the other groups. In the UK subjects, there were no differences in cystatin C between HNF1A-MODY and the other diabetic subgroups, except HNF1B-MODY. In UK HNF1A-MODY, cystatin C-based GFR estimate was higher than the creatinine-based one (p < 0.0001). Concluding, we could not confirm our hypothesis (supported by the Polish results) that cystatin C level is altered by HNF1A mutations; thus, it cannot be used as a biomarker for HNF1A-MODY. In HNF1A-MODY, the cystatin C-based GFR estimate is higher than the creatinine-based one.Cystatin C is a marker of glomerular filtration rate (GFR). Its level is influenced, among the others, by CRP whose concentration is decreased in HNF1A-MODY. We hypothesized that cystatin C level might be altered in HNF1A-MODY. We aimed to evaluate cystatin C in HNF1A-MODY both as a diagnostic marker and as a method of assessing GFR. We initially examined 51 HNF1A-MODY patients, 56 subjects with type 1 diabetes (T1DM), 39 with type 2 diabetes (T2DM) and 43 non-diabetic individuals (ND) from Poland. Subjects from two UK centres were used as replication panels: including 215 HNF1A-MODY, 203 T2DM, 39 HNF4A-MODY, 170 GCK-MODY, 17 HNF1B-MODY and 58 T1DM patients. The data were analysed with additive models, adjusting for gender, age, BMI and estimated GFR (creatinine). In the Polish subjects, adjusted cystatin C level in HNF1A-MODY was lower compared with T1DM, T2DM and ND ( HNF1A, encoding hepatocyte nuclear factor-1alpha, are a common cause of maturity-onset diabetes of the young (MODY) in Europeans [Cystatin C is a low-molecular-weight protein produced at a constant rate in all nucleated cells; it is freely filtered in the renal glomeruli, reabsorbed and catabolized in the proximal tubules . Cystatiuropeans , but noturopeans . This fouropeans . Making uropeans . CurrentHNF1A mutations. Additional justification for this study has come from the fact that the clinical picture of HNF1A-MODY includes several renal phenotypes, including tubulopathy with generalized aminoaciduria and glycosuria [The recently published report described lower hsCRP levels in HNF1A-MODY . Interesycosuria –13. The In an initial study, we included 51 HNF1A-MODY patients, 56 subjects with type 1 diabetes mellitus (T1DM), 41 with type 2 diabetes mellitus (T2DM) and 43 non-diabetic individuals (ND) from Poland. Subjects from two UK centres were used as replication panels. The Oxford subjects consisted of 36 HNF1A-MODY cases and 36 young-onset T2DM (diagnosed ≤45 years of age) matched for gender, current age and serum creatinine. The Exeter subjects consisted of 179 HNF1A-MODY, 167 T2DM, 39 HNF4A-MODY, 170 GCK-MODY, 17 HNF1B-MODY and 58 T1DM patients. In the UK (Oxford) group, one HNF1A-MODY subject and 5 T2DM subjects were of non-European origin. All the other individuals were European Caucasians.In all three centres, serum creatinine was analysed using a Jaffe method with calibration aligned to IDMS. In Poland and Exeter, the measurements were done using the Roche P800 analyser , while in Oxford, the ADVIA 2400 device  was used. Polish samples were analysed for cystatin C using a particle-enhanced assay nephelometric immunoassay (PENIA) with N-Latex cystatin C kit on a BNNII analyser . The UK samples were analysed using particle-enhanced turbidimetric immunoassay (PETIA) using either the ADVIA 2400 analyser (Oxford) or the P800 modular system (Exeter). HsCRP was analysed on the Polish samples using immunoturbidimetry.p values <0.05 were considered significant. The statistical analysis was performed with SAS 9.2.We estimated GFR from creatinine with the CKD-EPI formula  and from2 [p = 0.95) and significantly lower than in the other diabetic groups (p < 0.0001 for both groups). Adjusting for differences in CKD-EPI eGFR, gender, age and BMI revealed a significantly lower cystatin C level in HNF1A-MODY compared with T1DM, T2DM and ND groups . With adjustment for eGFR only and with adjustment to HbA1c among diabetic groups, the pattern of mean cystatin C levels and statistical significance of differences remained unchanged.The study groups’ characteristics are shown in Table 2 ; there w2 less, 95% CI: −7.3–4.6), nor in T2DM . In T1DM, the CKD-EPI estimate was 12.5 ml/min/1.73 m2 higher , while in the HNF1A-MODY group the difference was in the opposite direction: the CKD-EPI estimate was 13.8 ml/min/1.73 m2 less than cystatin C-based GFR . These results were not altered by adjusting for possible confounders.Using a linear model, instead of additive as the sensitivity analysis, did not change the conclusions or statistical significance, nor did exclusion of subjects with CKD. To further investigate the observed reduced cystatin C levels in HNF1A-MODY, we compared the patients’ GFR estimated with the CKD-EPI formula with the GFR estimated from cystatin C . CRP was a significant predictor of cystatin C concentration (p < 0.0001). The pattern of differences in creatinine and cystatin C GFR estimates was in line with the pattern of mean serum CRP concentrations measured in the groups. Specifically, in the groups with the highest (T1DM) and the lowest (HNF1A-MODY) CRP values, we observed, respectively, a reduced and an increased cystatin C-based GFR estimate, as compared with creatinine-based CKD-EPI estimates.Concordant with recent publications , HNF1A-Mp = 0.8), either before or after adjusting for covariates (as in Polish cohort). In both Oxford subject groups, we observed similar large differences between cystatin C-based and creatinine-based GFR estimates: the cystatin C-based measure was 27.4 ml/min/1.73 m2  higher in HNF1A-MODY and 25.8 ml/min/1.73 m2  higher in T2DM.We subsequently attempted to replicate these findings in the UK subjects. We observed no differences between cystatin C concentrations in HNF1A-MODY and T2DM patients from Oxford (p = 0.3), T2DM (p = 0.1), GCK-MODY (p = 0.4) and HNF4A-MODY (p = 0.9). The HNF1B-MODY subjects had significantly higher cystatin C, by 0.333 mg/l (95% CI: 0.178–0.488), compared with HNF1A-MODY (p < 0.0001). In the HNF1A-MODY group, the cystatin C-based GFR estimate was higher than the creatinine-based one by 5.3 ml/min/1.73 m2 . Thus, this finding was consistent in all three HNF1A-MODY groups. A higher cystatin C-based GFR estimate was also observed in the T1DM subjects recruited through Exeter; the difference between GFR estimates was 9.6 ml/min/1.73 m2 . There was no statistically significant difference in the GFR estimates in the HNF4A-MODY (p = 0.5), GCK-MODY (p = 0.5) and T2DM (p = 0.8) groups, while in the HNF1B-MODY subjects, the cystatin C-based GFR estimate was lower (p = 0.0009), consistent with the significantly higher cystatin C concentration observed in this subgroup.In the Exeter subjects, there were no significant differences in cystatin C  between HNF1A-MODY and T1DM  that cystatin C level is altered by


Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA) may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70 It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass and the maintenance of muscle mass contributes significantly to health and issues associated with the quality of life s6k). Active p70s6k can, in-turn, promote an increase in the helicase activity of eIF4A, and thus, provide an additional stimulus for the initiation of protein synthesis One mechanism that has been widely implicated in the regulation of protein synthesis involves signaling through the mammalian target of rapamycin (mTOR) mTOR is a Serine/Threonine kinase that is typically found in characteristically distinct multi-protein complexes. For example, the mTOR complex 1 (mTORC1) contains unique accessory proteins such as the regulatory-associated protein of mTOR (Raptor). On the other hand, the mTOR complex 2 (mTORC2) contains a protein called Rictor, but not Raptor. Furthermore, signaling by mTORC1, unlike mTORC2, is highly sensitive to inhibition by the drug rapamycin et al. (2011) recently demonstrated that mechanical overload induces an increase in the phosphorylation of TSC2 on the S664 residue, which is a site that can be phosphorylated by ERK, and potentially promotes the activation of mTOR signaling. Combined, all of these studies suggest that signaling through ERK could be an important part of the mechanism via which mechanical signals activate mTOR signaling.One mechanism that has been implicated in the mechanical activation of mTOR signaling, but has not yet been directly tested, involves signaling by the extracellular regulated kinase (ERK) A potential role of ERK is also highlighted by previous studies which suggest that phosphatidic acid (PA) may be involved in the mechanical activation of mTOR signaling. Specifically, work from our lab has shown that mechanical stimuli induce an increase in the concentration of PA in-vivo administration of MEK/ERK inhibitors, such as U0126, have hampered a direct investigation into this potentially important mechanism ex-vivo model of mechanical stimulation in which ERK inhibition was fully effective. Combined, our results demonstrate that the mechanical activation of mTOR signaling, and protein synthesis, occur through an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR.Taken together, the aforementioned studies suggest that ERK could play an important role in the mechanical activation of mTOR signaling. However, limitations with the sn-glycero-3-phosphate (C8 PA), egg L-α-phosphatidic acid (egg PA), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) were purchased from Avanti Lipids . For cell stimulation experiments, C8 PA and egg PA were prepared by drying lipids under nitrogen gas and dissolving in PBS with 3 min of water bath sonication at concentration of 0.6 mM and 6 mM respectively. For in-vitro stimulation experiments, C8 PA vesicles (50% C8 PA +50% PC) or PC vesicles (100% PC) were prepared by drying lipids as described above and dissolving in vesicle buffer (150 mM NaCl and 10 mM Tris pH 8.0) with 5 min of water bath sonication at a concentration of 6 mM. U0126 was purchased from Cell Signaling  and dissolved in DMSO at concentration of 50 mM or 20 mM before adding to the ex-vivo organ culture media or cell culture media, respectively. Rapamycin was purchased from LC laboratories  and dissolved in DMSO at a concentration of 150 µM before adding to ex-vivo organ culture media. Acetic acid, ethyl acetate, trimethylpentane and trichloroacetic acid were purchased from Fisher Chemical . 3H-Myristic acid and 3H-Phenylalanine were purchased from Perkin Elmer . Rabbit anti-phospho-p70s6k (Thr389) and anti-total 4E-BP1 , anti-total p70s6k, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-total ERK1/2, anti-phospho-S6 (Ser240/244), anti-phospho-S6 (Ser235/236), anti-total S6, anti-phopho-4E-BP1 (Thr37/46), anti-phospho-4E-BP1 (Ser65) , anti-total 4E-BP1 (53H11), anti-phospho-PKB (Ser473), and anti-total PKB were purchased from Cell Signaling .1,2-dioctanoyl-Eight- to ten-week-old inbred or outbred  FVB/N male mice were used for all experiments. For inbred mice, wild type FVB/N mice were bred with hemizygotic FVB/N mice that contain human skeletal actin promoter-driven expression of a FLAG-tagged rapamycin-resistant (Ser2035Thr) mutant of mTOR ex-vivo organ culture system which consisted of a refined myograph apparatus  and an organ culture bath as previously described 2 and 5% CO2 gassing, and fresh media was added to the bath at 30 min intervals.Mouse extensor digitorum longus (EDL) muscles were placed in an Lo) of EDL muscles was obtained at 13.5 mN). The muscles were then subjected to intermittent 15% passive stretch as a source of mechanical stimulation or held static at Lo as a control condition as previously described For mechanical stimulation, the EDL muscles were connected to the lever arms of a force transducer and micromanipulator, and then placed in the organ culture bath. The length of the EDL muscles were then adjusted until a passive tension of 13.5 mN was obtained  and 10% fetal bovine serum . These cells were plated on 6-well dishes and grown to confluence. Upon confluence, all cells were serum-starved in the absence of any antibiotics and antimycotics overnight before being subjected to experimental treatments. Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.For stimulation experiments, wild type C2C12 myoblasts were purchased from ATCC  and cultured in growth media consisting of high glucose DMEM supplemented with antibiotics and antimycotics  and equal amounts of protein from each sample were dissolved in Laemmli buffer before being subjected to SDS-PAGE. Following the electrophoretic separation, proteins were transferred to a PVDF membrane, blocked with 5% milk in TBST  for 1 h, and probed with rabbit primary antibodies overnight at 4°C. The membranes were then washed for 30 min in TBST and incubated with a peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. Following 30 min of washing in TBST, the blots were developed on film using ECL [Pierce  for Regular ECL and Amersham  for ECL Plus]. Once the appropriate image was captured, the membranes were stained with Coomassie Blue to verify equal loading througout all lanes. Densitometric measurements of each blot were carried out using ImageJ (NIH).Collected muscles were homogenized with a Polytron in an ice-cold lysis buffer  were made by dividing the amount of radioactivity in the PA spot by the amount of radioactivity in the total lipid extract.The method for measuring the concentration of PA in skeletal muscles Mouse wild type C2C12 myoblasts were cultured as described above. C2C12 myoblasts stably expressing FLAG-tagged mTOR were obtained from Dr. Jie Chen  and maintained in DMEM containing 0.2 mg/ml G418 (HyClone). G418 was not included in the media after the cells had been plated for the experiments. Upon confluence, cells were serum-starved overnight and subsequently collected in ice cold CHAPS lysis buffer . Fresh cell lysates were centrifuged at 2500 g for 5 min, and 400 µg of protein from the supernatant was diluted to a volume of 0.5 mL with fresh ice cold CHAPS lysis buffer. Samples were immunoprecipitated for the FLAG tag by incubating with 10 µL of EZview red ANTI-FLAG M2 agarose affinity gel beads  and gently rocking at 4°C for 3 h. Following the incubation, the beads were pelleted by centrifugation at 500 g for 30 sec and washed 3 times with fresh ice cold Wash buffer . The beads were then washed 2 times with Kinase Wash buffer (25 mM HEPES pH 7.4 and 20 mM KCl).Materials section. The vesicles were diluted to 150 µM in 1.5-fold concentrated mTOR kinase assay buffer (see below), and 10 µL of this solution was incubated with the immunoprecipitates at 30°C for 15 min. Then, mTOR kinase activity towards purified GST-tagged p70 For lipids stimulation, C8 PA or PC vesicles were prepared as described in the post hoc analysis. Differences between groups were considered significant when P≤0.05. All statistical analyses were performed on SigmaStat software .All values are expressed as means (+ SEM in graphs). Statistical significance was determined by using ANOVA, followed by Student–Newman–Keuls ex-vivo organ culture system in which mouse EDL muscles were mechanically stimulated with intermittent passive stretch. In this model, we determined that both 15 and 90 min mechanical stimulation was sufficient to induce an increase in the phosphorylation of ERK1 (T202/Y204). Furthermore, in this system, the MEK/ERK inhibitor U0126 was fully capable of inhibiting signaling through ERK , and 4E-BP1, are commonly used as markers of mTOR activity. For example, the T389 residue on p70s6k is a widely accepted mTOR-specific phosphorylation site s6k contain the consensus “S/T-P” sequence which can be phosphorylated by proline-directed kinases such as ERK Changes in the phosphorylation status of molecules such as p70s6k on residues such as S235/236 and S240/244 As mentioned above, the phosphorylation status of S6 is also commonly used as a marker of mTOR activity, and previous studies have shown that S6 is predominantly phosphorylated by p70In order to further clarify the role of ERK in the mechanical activation of mTOR signaling, we measured the phosphorylation status of 4E-BP1. For example, we measured changes in gel mobility as a marker of global changes in 4E-BP1 phosphorylation s6k, S6, and 4E-BP1, are commonly used as markers of mTOR activity. However, many of the phosphorylation sites on these molecules can be regulated by both mTOR-dependent and mTOR-independent events s6k T389 and 4E-BP1 S64 phosphorylation, suggesting that these events occur through an mTOR-dependent mechanism. Interestingly, the mechanically-induced decrease in total 4E-BP1 was also eliminated by rapamycin, which suggests that the loss of 4E-BP1 may be a previously unrecognized mTOR-dependent signaling event. On the other hand, rapamycin did not prevent mechanical stimulation from inducing an increase in the phosphorylation of the p70s6k T421/S424, S6 S235/236 and S6 S240/244 residues. Thus, it can be concluded that rapamycin-insensitive/mTOR-independent mechanism(s) are at least partially responsible for the mechanically-induced phosphorylation of these residues.As previously noted, changes in the phosphorylation status of molecules, such as p70s6k T389 and 4E-BP1 S64 phosphorylation, and the decrease in total 4E-BP1, were all effectively rescued from the inhibitory actions of rapamycin. Therefore, it can be concluded that these events are indeed valid markers of mechanically-induced signaling by mTOR.Like all pharmacological inhibitors, rapamycin can potentially exert non-specific (mTOR-independent) actions. For example, it has been shown that rapamycin can bind and sequester the FKBP12 protein and this can interfere with the role that FKBP12 plays in the function of the ryanodine receptor and signaling by members of the transforming growth factor-β superfamily Previous studies have indicated that signaling through mTOR is necessary for a mechanically-induced increase in the rate of protein synthesis, and that the activation of mTOR signaling is sufficient to induce protein synthesis 3H-myristic acid labeled PA.Previous studies have suggested that a mechanically-induced increase in PA is necessary for the activation of mTOR signaling s6k T389 and 4E-BP1 S64 phosphorylation, or the loss of total 4EBP1 . However, a more robust increase in signaling through ERK has been observed with other types of mechanical stimulation. For example, Martineau and Gardiner 2001, demonstrated that concentric, isometric, and eccentric contractions produce a 3-, 4-, and 5-fold increase in signaling through ERK, respectively et al., 2011 In this study we employed an in-vitro. Furthermore, like mechanical stimuli, we found that PA induces mTOR signaling through an ERK-independent mechanism. All of these observations are consistent with a model in which mechanical stimuli induce an increase in PA, and the newly formed PA then binds and activates mTOR. Although intriguing, additional studies will be needed to test the validity of this concept.To date, numerous studies have attempted to identify the molecular mechanisms that are involved in the mechanical activation of mTOR signaling s6k T389 phosphorylation is fully dependent on mTOR. Conversely, we found that other commonly used markers of mTOR signaling, such as S6 S235/236 and S6 S240/244 phosphorylation, were at least partially induced via a rapamycin-insensitive/mTOR-independent mechanism. Consistent with these results, another recent study also demonstrated that rapamycin does not block mechanical overload-induced increases in S6 S235/236 and S6 S240/244 phosphorylation s6k1 and p70s6k2 double knockout mice demonstrated that S6 S240/244 phosphorylation requires p70s6k activity s6k, the activation of unrecognized S6 kinase(s), or an inhibition of S6 phosphatases. Furthermore, our observations illustrate that caution should be used when interpreting changes in S6 S235/236 or S6 S240/244 phosphorylation as markers of mTOR signaling.In this study, we also used RR-mTOR mice to identify valid markers of mechanically-induced mTOR signaling. For example, the RR-mTOR mice enabled us to demonstrate that a mechanically-induced increase in p70s6k T389 and 4E-BP1 T36/45 residues s6k T389 and 4E-BP1 T36/45 phosphorylation s6k T389 phosphorylation, in conjunction with no change in 4E-BP1 T36/45 phosphorylation, was highly unexpected. Although the reason for this disparity is not known, a recent study by Yip et al. 2010 may have revealed some important clues. Specifically, Yip et al. demonstrated that rapamycin inhibits the ability of immunopurified mTOR to phosphorylate p70s6k T389 and 4E-BP1 T36/45 s6k T389. Yet, in the absence of raptor, the ability of mTOR to phosphorylate p70s6k T389 was still sensitive to inhibition by rapamycin. In other words, it appears that there is a rapamycin-sensitive pool of mTOR that does not involve the classical raptor-associated mTORC1 complex. Similar to the results we observed with mechanical stimulation, this uncharacterized rapamycin-sensitive pool of mTOR can phosphorylate p70s6k T389, but not 4E-BP1 T36/45. Thus, it is very tempting to speculate that mechanical stimuli activate a unique rapamycin-sensitive pool of mTOR that is distinct from mTORC1.The RR-mTOR mice also enabled us to demonstrate that mTOR is necessary for mechanical stimulation to induce an increase in 4E-BP1 S64 phosphorylation, and a decrease in total 4E-BP1. However, much to our surprise, mechanical stimulation did not alter 4E-BP1 T36/45 phosphorylation. This observation was surprising because numerous studies have shown that, when activated, the rapamycin-sensitive mTORC1 can directly phosphorylate both p70As mentioned in the introduction, signaling through mTOR has been widely implicated in the regulation of protein synthesis. For example, the mTOR kinase inhibitor (Torin 1) induces a greater than 60% reduction in the rate of protein synthesis when added to wild-type mouse embryonic fibroblasts (MEF). However, Torin 1 has essentially no effect on the rate of protein synthesis when added to MEFs that are deficient of 4E-BP1 and 4E-BP2 in-vivo eccentric contractions as described in In general, mTOR is thought to control the function of the 4E-BPs by inducing changes in the phosphorylation state of the protein. For example, mTOR can directly phosphorylate the 36/45 residues of 4E-BP1, and phosphorylation on these residues allows 4E-BP1 to become further phosphorylated on residues such as S64 In summary, the results from this study demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Since signaling through mTOR is necessary for a mechanically-induced increase in protein synthesis, and ultimately growth, these findings should help advance our understanding of how mechanical signals are converted into the molecular events that regulate skeletal muscle mass.Figure S1Mechanical stimulation does not increase protein synthesis in muscles incubated with KHB media. EDL muscles were held at Lo and pre-incubated for 30 min with KHB media containing 0.1% DMSO, 1X MEM amino acids and 25 mM glucose. The muscles were then subjected to 90 min of stretch or control conditions, and protein synthesis rates were measured during the final 30 min. All values are presented as the mean + SEM (n = 4−6 per group).(TIF)Click here for additional data file.Figure S2Mechanical stimulation activates mTOR signaling via an ERK-independent mechanism in DMEM media. EDL muscles were held at Lo and pre-incubated with DMEM media containing 50 µM U0126 (U0126+) or the vehicle  for 30 min. The muscles were then subjected to 90 min of stretch or control conditions. Muscles were collected at the end of the 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70 and total 4EBP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples . Note: total 4E-BP1 was measured with two different antibodies, i) a monoclonal antibody from Cell Signaling that was raised against a peptide surrounding the Ser112 residues (C.S.), and ii) a polyclonal antibody from Santa Cruz that was raised against full length 4E-BP1 as the antigenic peptide (S.C.). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.(TIF)Click here for additional data file.Figure S3Exogenous phosphatidic acid induces protein synthesis via an ERK-dependent mechanism. C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 or the vehicle (DMSO) for 30 min, followed by 60 min stimulation with 30 µM C8 PA or the vehicle (PBS). Protein synthesis rates were measured during the final 30 min and expressed as a percentage of the vehicle control values. All values are presented as the mean + SEM and were obtained from five independent experiments (n = 9−11 per group). *Significantly different from the drug-matched control group. †Significantly different from the stimulation-matched vehicle group, P≤0.05.(TIF)Click here for additional data file.