Bombyx mori L. (Lepidoptera: Bombycidae) caspase family genes are poorly known. This paper focuses on the morphology, mitochondrial membrane potential, and expression profiles of two novel B. mori caspase family genes (ice-2 and ice-5) in 3 µM hydrogen peroxide (H2O2) damaged B. mori cells, which were separated from the ovary of B. mori. In addition, comparisons were made between damage caused by H2O2 and by ultraviolet (UV) irradiation. The results showed that the potential change of the mitochondrial membrane occurred at 0.5 h after H2O2 stimulation, which was sooner than occurred in the UV treated model where the obvious decrease appeared at 6 h after stimulation. In addition, the total change in the potential of the mitochondrial membrane in H2O2 treated B. mori cells was larger than with UV treated cells during the whole process. Analysis of fluorescent quantitative real-time PCR demonstrated that ice-2 and ice-5 might be involved in both H2O2 and UV-induced apoptosis in B. mori cells. Notably, after exposure to H2O2, the expression patterns of ice-5 were remarkably higher than those of ice-2, while the result was the opposite after exposure to UV irradiation. The data indicate that apoptosis induced by H2O2 was directly related to the mitochondrial pathway. The two isoforms of B. mori ice may play different roles in the mitochondrion associated apoptotic pathway in B. mori cells, and the apoptotic pathway in H2O2 induced B. mori cells is different from the UV induced apoptotic pathway.Caspase family proteins play important roles in different stages of the apoptotic pathway. To date, however, functions of  Caenorhabditis elegans . In a previous study (ice-2 and ice-5 were cloned with an open reading frame of 852 and 936 base pairs (bps), respectively.As a member of the caspase (cys-teiny-laspartate specific proteinase) family, interleukin -1 beta-converting enzyme (ICE) was discovered in mammals and named caspase-1. It is considered the initiator in caspase-dependent apoptosis. ICE was identified as a CED-3-like protein in  elegans . In lepi process . Accordius study  ice-2 an2O2 is a reactive oxygen species. In general, reactive oxygen species are harmful to living organisms because they tend to cause oxidative damage to proteins, nucleic acids, and lipids . Meanwhile, time course transcriptional profiles of the two genes were investigated by quantitative realtime PCR. This report will provide new insight into the function of ICEs in insects. Additionally, damage caused by H2O2 and UV irradiation were compared in this paper and may provide insight into the role of insect ICEs during the apoptosis processes.This study attempted to characterize the genes of B. mori ovary-derived cells that were a gift of Dr. Xiangfu Wu  were cultured in TC-100 insect cell culture medium  supplemented with 10% fetal bovine serum at 27° C. H2O2 was applied to the B. mori cells, which then were plated at a density of 2 × 106 cells in 6-well plates . They were incubated for 3–5 days at 27° C, and then used for further studies.B. mori cells by exposure to different concentrations (0.09 – 90 µM) of H2O2, and the median lethal dose (LD50) was calculated. While incubating at the LD50 H2O2 concentration, B. mori cells were observed microscopically at specified intervals for the appearance of apoptotic bodies, and were collected at regular intervals.Apoptosis was induced in 2). The total dosage was measured by a radiometer  fitted with a UV detector. At the LD50 H2O2 concentration, LD50, B. mori cells were observed microscopically at specified intervals for the appearance of apoptotic bodies, and were collected at regular intervals.The cells, with a very thin layer of phosphate buffered saline were irradiated for 20 s under UVA and UVB lamps at different UV doses (50 - 5 mJ/cmwww.moleculardevices.com). Mortality = 1-viability.The 3--2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect mortality and was carried out according to Fornelli et al. . Five mgB. mori cells by using the lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodine (JC-1) (2 (bottom scanning).Change in the potential of the mitochondrial membrane was assessed in live e (JC-1) . For quawww.promega.com). The concentration of the RNA was assessed using the Genspec III spectrophotometer , and the integrity of the RNA was assessed by running 2 µl of RNA on a 1% ethidium bromide/agarose gel. The RNA was stored at -70° C until needed.Total RNA was extracted from the collected cells using Trizol (Invitrogen) according to the manufacturer's protocol. Contaminated genomic DNA was removed by Rnase-free Dnase I . The thermal cycling profiles were as follows: 65° C for 5 min, 37° C for 60 min, and 75° C for 5 min. The resultant cDNA was stored at -20° C until needed.ice-2, ice-5 and B. mori actin were selected based on the sequences available in GenBank. Primers were designed for specific detection . Primers for B. mori actin A3 (Forward: 5′ atccagcagctccctcga gaagtc t 3′ and Reverse: 5′ acaatggagggacca gactcgtcgt 3′) were used as an endogenous reference gene in real-time PCR.Primers used for the real-time PCR amplification of ice-2 and ice-5 in treated B. mori cells in the sequence detection system . Duplicates of 0.5 µl cDNA from each reverse transcription reaction were used as templates. The reactions were performed in a total volume of 50 µl using SYBR premix EXTaqTM perfect realtime kit  as recommended by the manufacturer. The following MX3000P thermocycling program was used: denaturation program (3 min at 95° C), amplification and quantification program repeated 40 times , and melting curve program (55° C to 95° C with a heating rate of 0.1° C/s).Real-time PCR amplifications were performed to examine the relative expression of ice-2 and ice-5 were calculated with the comparative Ct (2 -ΔΔCt) method. Means and standard errors for each time-point were obtained from the averages of three independent sample sets.Relative expression levels of www.spss.com) with one-way ANOVA and one-sample T test. The p value lower than 0.05 was considered as significant.Data are presented as the mean ± SD or mean ± SE of results of two or three separate experiments, as specified in the figure legends. Statistical significance was calculated  of H2O2, and the LD50 value was calculated using the MTT assay. The same test was repeated with UV irradiation. 2O2 was 3 µM because mortality (49.074%) of 3 µM-treated B. mori cells was nearest to LD50. The best dose of UV irradiation was 20 mJ/cm2, with a mortality rate of 45.961%, which was the nearest to LD50.Apoptosis was induced in B. mori cells were observed after H2O2 stimulation at regular intervals from 0 to 12 h. As time passed, the morphology of the cells changed. However, in the first 4 h after stimulation, there were a few cells that had different morphology from the normal cells  was more obvious than that of the UV treated cells (4.938)  . In H2O2ed cells .2O2 might relate to the intrinsic apoptotic pathway via effects on the mitochondria. The peak levels of ice-2 and ice-5 were reached when the cellular morphology was still unchanged but the mitochondrial membrane potential had already changed considerably (B. mori ice-2 and ice-5 might be related to the release of cytochrome c from the mitochondria. Later, at 5 h after stimulation, changes in all the data were obvious. First, cell membranes were triggered to wrinkle, and cells became smaller than the ordinary cells. At the same time, the mitochondrial membrane potential steadily declined, beyond the dramatic decrease during the first 5 h. There was also another increase in the expression of ice-2 and ice-5. In Spodoptera frugiperda cells, oxidant treatments resulted in the release of cytochrome c followed by the activation of caspase-3 (B. mori ICEs might be regulated by H2O2, and related to the dysfunction of mitochondria, ice-2 and ice-5 may also be initiators associated with mitochondria initially, and then be effectors following the dysfunction of mitochondria in H2O2 induced apoptosis.As previously reported, the decrease of mitochondrial membrane potential started at the very beginning of the treatment and preceded the morphological change of the cells. This implies that apoptosis induced by Hiderably , Table 2aspase-3 . Therefoice-2 and ice-5 were different from each other by just one exon implied that different mRNAs are present. This is likely related to the different patterns in their expression profiles. From 0 to 0.5 h after exposure to H2O2, while the level of ice-2 increased from low to high, the level of ice-5 increased from being undetectable to the highest level (ice-2 rose to its highest level, and ice-5 reached its second peak, suggesting that ice-5 may play a more active role in the early phase of H2O2-induced apoptosis than ice-2, and that they may have complementary functions, ice-2 and ice-5 might induce their own expression of in the later phases of apoptosis.The fact that the genes of st level . Then, aice-2 and ice-5 decreased significantly at 1 h after H2O2 stimulation, and the level of ice-2 remained low from 1 to 4 h after H2O2 stimulation. In contrast, the level of ice-5 fluctuated from low to a medium during this period. This was quite different from the profile of UV induced apoptosis (ice-5 was almost undetectable. This difference may have resulted in the changing morphology of B. mori cells at 5 h after stimulation. The unique expression patterns of ice-2 and ice-5 suggest that the single exon difference between them may be the reason for the unique role of ice-5 in the apoptotic pathway.Based on the expression profiles, the levels of both poptosis . During 2O2 treated cells is about 3 times more than the reduction in fluorescence ratio of UV treated cells. This suggests that H2O2-induced damage led to a more serious loss in the potential of the mitochondrial membrane (ice-2 and ice-5 were less active in UV stimulated cells.In addition, the total reduction in fluorescence ratio of Hmembrane . This mamembrane . When thice-2 and ice-5 synchronal expression profiles indicate that activation of ice-2 and ice-5 may be related to mitochondrial dysfunction after H2O2-induced damage and that ice-2 and ice-5 might cooperate in the early phases of both H2O2 and UV induced apoptosis in a B. mori cell line. The comparison between relative expression profiles of H2O2 and UV-induced apoptosis suggests that the absence in ice-2 of an 84bp exon that exists in ice-5 might be the reason for lower activity of ice-2 than of ice-5 in the H2O2 induced apoptosis pathway. Because UV irradiation not only induces the generation of OH and H2O2 (2O2-induced apoptosis. This phenomenon would occur uniquely in UV irradiationinduced apoptosis and is a topic for further study.In conclusion, and H2O2 , but als


Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called “reverse signalling”. In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells. We found that bone marrow-derived macrophages were indeed stimulated by TACI-Fc and BAFFR-Fc, but not by an irrelevant decoy receptor, Fn14-Fc, that target the TNF family ligand TWEAK. As confirmed in ligand-deficient cells, this activation was however independent of BAFF or APRIL expression, but dependent on the presence of protein aggregates that induced Fc receptor-mediated signalling.Primary mouse macrophages (BMDMs) exposed to endotoxin-free TACI-Fc or BAFFR-Fc displayed pronounced phosphorylation of ERK within 5 min of stimulation. In addition, Akt and IκBα were also phosphorylated, but neither TACI-Fc nor BAFFR-Fc appeared to significantly alter the p52 to p100 ratio, a hallmark of the alternative NF-κB pathway . None ofWe next tested the human monocytic cell line THP1 and the human macrophage cell line U937 cells for their responses to BAFFR-Fc and TACI-Fc. Similar to what we observed in mouse macrophages, THP-1 cells responded to BAFFR-Fc and TACI-Fc, but not to Fn14-Fc, with increased ERK and Akt phosphorylation , and somTaken together these results show that TACI-Fc and BAFFR-Fc, but not Fn14-Fc, can signal in myeloid cells of both mouse and human origin. Because of the Fn14-Fc control, we considered an Fc-mediated effect unlikely and instead favoured the hypothesis that TACI-Fc and BAFFR-Fc might exert their effects by inducing BAFF reverse signalling in these cells, all of which are known to express BAFF Stimulation of HEK-293 cells stably expressing BAFF at their surface −/−, BAFF−/− mice or from mice expressing an excess of non-cleavable BAFF . C57BL/6 WT, BAFFE. coli K14 ultrapure LPS was from Invivogen. His-tagged BAFF 60-mer was from Adipogen. Antibodies for western blot were: mouse IgG1 anti-phospho-p42/44 (P-ERK) and mouse IgG1 anti-tubulin from Sigma, mouse IgG1 anti-phospho-IκBα, mouse IgG2b anti-phospho-Akt Ser473, rabbit anti-IκBα rabbit anti p100/p52, rabbit anti-Akt from Cell signalling, mouse IgG1 anti CD3 ζ and rabbit anti-pospho-JNK from Biosource and goat anti-ERK1 from Santa-Cruz.293 wt (ATCC CRL 1573) and 293-hBAFF full-544 cells were cultured in DMEM:F12 (1∶1 v/v) supplemented with 2% foetal calf serum 4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH7.3), washed in complete medium and seeded at 106 cells/ml in BMDM differentiation medium  in petri dishes for bacteriology. Cells were cultured at 37°C, 5% CO2 for 6 days, with addition on day 3 of 500 µl of fresh medium per ml of culture. Cells were then detached in PBS, 20 mM Hepes, 5 mM EDTA , plated at 106 cells/condition in 500 µl of RPMI 10% FCS in 48 well-plates, and left to attach overnight before stimulation.BMDMs were obtained as described 6 BMDMs or BMDCs were stimulated for 0, 5, 10, 30, 60 or 120 min with various receptors-Fc at 5 µg/ml or with LPS at 50 ng/ml in 500 µl of RPMI 10% FCS. 3×105 HEK-293 wt or HEK-293-BAFF cells were stimulated with hBAFFR-Fc at 5 µg/ml in 500 µl for the indicated times, or with 60 ng/ml of PMA. 4×104 THP1 or U937 cells in 200 µl of RPMI 10% FCS in flat-bottom 96-well plates were stimulated for 0, 10, 30, 120 min with various receptors-Fc at 5 µg/ml or LPS at 50 ng/ml in 200 µl of RPMI 10% medium. At the end of the stimulation, medium was rapidly discarded and cells lysed immediately in 60 µl of 3 times concentrated, reducing SDS-PAGE sample buffer (30 µl for a well of a 96-well plate). Non adherent cells were first centrifuged for 5 min at 400×g. Extracts were transferred to 1.5 ml microtubes using wide-opening tips, heated for 5 min at 95°C and sonicated for 15 min in an ice-cold sonicating bath.105 for BMDM and BMDC, 105 for HEK-293 cells and 4×104 for THP1 and U937 cells. Western blots were revealed with the antibodies indicated in the figures, at dilutions indicated in SDS-PAGE and Western blot were performed according to standard procedures, using the following number of cell equivalents per lane: 3×10Purified receptors-Fc were loaded in a volume of 400 µl on a Superdex-200 column  eluted in PBS with online absorbance monitoring at 280 nm and 1 ml fraction collection. Fractions of interest were filtered at 0.22 µm and concentration was calculated from absorbance at 280 nm using extinction coefficients of 1.328 for TACI-Fc and atacicept and 1.077 for BAFFR-Fc.The cytotoxicity assay using BAFFR:Fas cells was performed as described for FasL on Jurkat cells Figure S1List of antibodies used in this study.(PDF)Click here for additional data file.


Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease. Mammalian sterile 20—like kinase 1 (Mst1) is an ubiquitously expressed serine/threonine kinase with a similarity to the Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, cell growth, organ size control, apoptosis and tumor suppression in mammals 175, Thr177, Thr183, Thr187, Ser327 and Thr387, of which, Thr183 and Thr187 appear to be essential for kinase activation Regulation of Mst1 appears to occur mainly at posttranslational levels. In addition to its activation by proteolytic cleavage, Mst1 autophosphorylation has been proposed to contribute to the kinase activation Recently, the physiological role of Mst1 in the cardiovascular system has begun to be explored. In cardiomyocytes, Mst1 is activated by pathological stimuli, such as hypoxia/reoxygenation in vitro and ischemia/reperfusion in vivo HEK293T and Ad293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) and were maintained at 37°C with 5% CO2. Cells were transiently transfected with FuGENE 6 (Roche Applied Science) using the manufacturer's protocol.To identify novel proteins that interact with Mst1, we screened a human heart cDNA library using human dominant-negative Mst1 (K59R) as bait and the MATCHMAKER GAL4 yeast 2-hybrid system 3 (Clontech Laboratories Inc) as previously described 2 and 95% N2 at 37°C for 12 hours. Following hypoxia exposure, cells were reoxygenated by placing cells in the normoxic culture medium for 24 hours 26]We obtained ventricles from 1-day-old Sprague-Dawley rats and isolated cardiac myocytes by digestion with trypsin-EDTA and type 2 collagenase as previously described HEK293T cells were transiently transfected with Mst1 and GAPDH cDNAs using FuGENE 6 as described by the manufacturer (Roche Applied Science). Both HEK293T cells and cardiomyocytes were lysed in the buffer containing 1% Nonidet P-40, 150 mmol/L NaCl, 50 mmol/L Tris (pH 8), 100 µmol/L EDTA, and protease inhibitors. Coimmunoprecipitation of Mst1 and GAPDH was performed essentially as described Freshly isolated neonatal rat cardiomyocytes were fixed and sequentially incubated with primary antibodies and appropriate fluorescent-labeled secondary antibodies. Images were visualized using an Olympus IX70 epifluorescence microscope as previously described in vitro, recombinant GAPDH (Abcam) was incubated with recombinant active Mst1 (Upstate) and [γ-32P]-ATP (PerkinElmer) in 1× kinase assay buffer  at 30 °C for indicated time points. The reaction was terminated by adding 1/2 volumes of 3× Laemmli sample buffer and incubated at 95 °C for 5 min, then examined by 15% SDS-PAGE and a autoradiography.To phosphorylate GAPDH 2, 1 mmol/L DTT, and 1 µCi [γ-32P]-ATP. Reactions were terminated by the addition of 2× SDS sample buffer, and then loaded to 15% SDS-PAGE and subjected to autoradiography. For Mst1 kinase assay in cardiomyocytes, cell homogenates (400 µg) were immunoprecipitated using anti-Mst1 antibody (BD Transduction Laboratories), and then incubated with 2 µg Histone H2B (Sigma) in 25 µL kinase assay buffer for 20 min at 30°C. Samples were subjected to SDS-PAGE and phosphorylation of histone H2B was detected by immunoblotting with anti-phospho-Histone H2B (Ser14) antibody (Millipore).Recombinant active Mst1 (Upstate) or immunoprecipitated Mst1 from HEK293T transfected with Myc-Mst1 cDNA in combination with either empty vector or Flag-GAPDH was incubated for 20 min at 30°C with 4 µg Myelin basic protein (MBP) (Upstate) in 25 µL kinase buffer 40 mmol/L HEPES-NaOH (pH 7.4), 20 mmol/L MgClPredesigned GAPDH siRNA and control siRNA were purchased from Ambion. Cardiomyocytes were transfected with Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) with target-specific siRNA (20 nmol/L) and control siRNA (20 nmol/L) in serum-free medium according to the recommendations of the manufacturer.2 banding, followed by dialysis against 20 mmol/L Tris-buffered saline with 10% glycerol. Titering was performed on Ad293 cells using Adeno-X Rapid Titer kit (Clontech) according to the instructions of the manufacturer.Adenoviruses harboring wild-type Mst1 (Ad-Mst1), dominant-negative Mst1 (Ad-DNMST), and GAPDH were made using AdMax (Microbix) as previously described C57BL/6 mice (20–25 g) were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (2.5 mg/kg) intraperitoneally. The mini-osmotic pumps  set to dispense 0.5 µl/hr for 14 days were filled with PBS or ISO (60 mg/kg/day) and pumps were implanted subcutaneously in the back through a sub-scapular incision, which was then closed using 4.0 silk suture (Ethicon). Mice were followed by echocardiogram prior to pump implantation and then weekly until the end of the 2 week period when mice developed cardiac hypertrophy. This study was reviewed and approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University. Tissue homogenates from normal heart and hypertrophied heart were then subjected to immunoprecipitation by using anti-Mst1 antibody as previously described Histone-associated DNA fragments were quantitated by the Cell Death Detection ELISA kit (Roche) t tests or ANOVA, as appropriate; a value of P<0.05 was considered statistically significant.Data are expressed as means ± SEM. The statistical significance of differences was assessed by Student's 6 clones, we identified 4 positive cDNAs encoding the C-terminal domain (aa 164–335) of GAPDH, suggesting that the C-terminal region of GAPDH may be sufficient to interact with Mst1 in yeast.In an effort to identify novel Mst1 binding proteins, a yeast 2-hybrid screening was performed with human dominant negative Mst1 (DN-Mst1) as bait in conjunction with a human heart cDNA library. After screening 2.4×10To determine whether the interaction between Mst1 and GAPDH occurs in mammalian cells, coimmunoprecipitation experiments were performed in HEK293T cells transfected with GAPDH and Mst1 expression vectors. Immunoprecipitation of FLAG-tagged GAPDH led to coimmunoprecipitation of Myc-tagged Mst1 when both proteins were cotransfected . As a coTo determine whether there is an endogenous interaction of GAPDH and Mst1 in cardiomyocytes, we performed immunoprecipitation with anti-Mst1 antibody using lysates obtained from neonatal rat ventricular cardiomyocytes (NRVMs). Indeed, GAPDH was co-precipitated with the anti-Mst1 antibody but not with the nonimmune IgG . The intTo further map the interaction domains of GAPDH and Mst1, we performed immunoprecipitation in HEK293 cells cotransfected with different deletion mutants of Mst1 and GAPDH. We first investigated the binding domains of GAPDH in Mst1. Mst1 contains an N-terminal kinase domain (aa 1–325), inhibitory domain (aa 326–294), and a C-terminal dimerization domain (aa 395–487) GAPDH contains two functional domains, namely, the NAD binding domain and the C-terminal catalytic domain 32P]ATP in an in vitro phosphorylation assay. Indeed, incubation of Mst1 with GAPDH induced a robust phosphorylation of GAPDH in a dose dependent manner  resulted in a robust phosphorylation of MBP, which was not significantly affected in the presence of GAPDH . MoreoveGAPDH has been shown to induce cell apoptosis through its nuclear translocation Because Mst1 has been characterized to induce cardiomyocyte apoptosis To further investigate the role of GAPDH in the regulation of Mst1 mediated cardiomyocytes, we used siRNA to knockdown the expression of GAPDH. Transfection of GAPDH siRNA (siGAPDH) reduced GAPDH expression by ∼80% in cardiomyocytes, as determined by both qRT-PCR  and westUsing the yeast 2-hybrid system and Mst1 as bait, we isolated several GAPDH cDNAs from a human heart cDNA library. The interaction of GAPDH with wild-type Mst1 was further supported by coimmunoprecipitation studies showing that in both cotransfected HEK293T cells and cardiomyocytes, GAPDH specifically interacts with Mst1. The interaction between GAPDH and Mst1 requires the kinase domain of Mst1 and it seems that the C-terminal catalytic domain of GAPDH mediates the binding of Mst1 in GAPDH. Interestingly, GAPDH is phosphorylated by Mst1 to a comparable extent as its known substrate MBP, suggesting that GAPDH is a good substrate of Mst1 at least in vitro. Furthermore, the functional consequence of this interaction was demonstrated by the ability of GAPDH to enhance Mst1 activity and Mst1-mediated apoptotic effects in cardiomyocytes. Indeed, inhibition of GAPDH expression attenuates the Mst1 activation and the Mst1 induced cardiomyocyte apoptosis in response to chelerythrine. These results suggest that the regulation of Mst1 activity by GAPDH may be an important determinant of cell survival in the heart.GAPDH is a key enzyme in the glycolytic pathway, which catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) to 1,3-biphosphoglycerate in the presence of NAD+ and inorganic phosphate The Mst1 signaling pathway plays an essential role in cell apoptosis The mechanism by which GAPDH activates Mst1 is not known, but certainly involves other components in Mst1 immunocomplexes, as such an increased Mst1 activity by GAPDH was only observed with immunoprecipitated Mst1 from HEK293 cells transfected with Mst1 cDNA. Indeed, the Mst1 kinase signaling cascade is tightly regulated by protein–protein interactions. Several proteins, including Rassf1, hWW45 In summary, we have found that GAPDH interacts with Mst1 kinase in cardiomyocytes. This interaction is specific and leads to a functional increase in Mst1 activity and Mst1 mediated cardiomyocyte apoptosis. These findings suggest that the GAPDH/Mst1 pathway may be an important therapeutic target for inhibiting myocardial apoptosis in ischemia-reperfusion injury and heart failure.


Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba.t-butyl)pyrazolopyrimidine) and its inactive analog, PP3 . Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells.Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo  pyrimidine) but not its inactive analog, PP3 , had detrimental effects on the growth of A. castellanii . Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier.Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl)-7-(A. castellanii proliferation, protease secretions and phagocytic properties. Conversely, invasion of Acanthamoeba by pathogenic bacteria was stimulated by Src kinase inhibition.For the first time, these findings demonstrated that Src kinase is involved in  Acanthamoeba represents 17 different groups, i.e., T1 – T17   in a T-75 tissue culture flask at 30 °C as previously described  but did not affect amoebic viability, as determined by Trypan blue exclusion test (0% effects). At micromolar concentrations of PP2 (50 μM), amoeba growth was abolished. In contrast, inactive analog Src kinase, PP3 affected neither growth nor viability of A. castellanii for any time point tested  , using T-test, paired, one tail distribution], although there was no difference at 10 μM (P = 0.21) , using T-test, paired, one tail distribution) , using T-test, paired, one tail distribution], although there was no difference at 10 μM (P = 0.077) , using T-test, paired, one tail distribution]  Figure . At 100 d Figure . In contA. castellanii protease secretion, conditioned medium (CM) was produced in the presence of Src kinase inhibitor, PP2, or its inactive analog, PP3 and analysed for protease activities. In the absence of any inhibitor, A. castellanii exhibited significant proteolytic activities . Next, to determine the effects of PP2 on A. castellanii-mediated HBMEC death, cytotoxicity assays were performed. In the absence of PP2, Acanthamoeba produced severe HBMEC cell cytotoxicity (up to 65%) within 24 h   , which was found to be similar in architecture to human cSrc and its orthologs . Putative AcSrc kinase shares considerable amino acid homology with its orthologs . However, it also has its unique N- and C-terminal regions which are not homologous to its orthologs but to a small hypothetical protein of the fungus Verticillium dahliae in NCBI database (data not shown). In the human cSrc kinases protein, phosphorylation of tyrosine at positions 419 and 530 (Y419 and Y530) marks the protein for active and inactive state, respectively [Acanthamoeba. The role of these conserved tyrosine residue Y530 is not clear in protist Src kinases, because the phosphorylation of MbSrc1 in Monosiga brevicolli at the tyrosine residue corresponding to Y530 of human cSrc kinase did not result in an inactivation of its kinase activity, unlike its human homologs [Acanthamoeba Src kinase, which might have a role in important cellular processes, such as growth and phagocytosis.A BLAST search of ris AAA297.1; 31% ectively . Of theshomologs , which mhomologs ,24 showeAcanthamoeba exhibits multifactorial virulence properties to produce damage of HBMEC, which constitute the blood–brain barrier, to produce encephalitis and corneal epithelial cells to induce keratitis [Acanthamoeba. The findings revealed that Src kinase inhibitor, PP2, but not its inactive analog, PP3, abolished A. castellanii proliferation. These findings indicate that Src kinases have a pivotal role in the growth of A. castellanii, which is consistent with some previous findings that showed that Src is an important signalling molecule involved in the growth of various eukaryotic cell types [castellanii suggest that they should be studied further.Recent studies have shown that eratitis . Here, wll types ,12. Src ll types -27. PhosA. castellanii use phagocytosis for food uptake (such as non-invasive E. coli K-12), whereas E. coli K1 , utilises A. castellanii as a reservoir and invades into the Acanthamoeba. Inhibition of Src kinase reduced A. castellanii phagocytosis of non-invasive K-12 with an increase in the concentration of PP2, while opposite effects were observed for invasive E. coli K1. A likely explanation is that E. coli K-12 is a food source for amoebae. In such situations, the process of bacterial uptake is most likely to be “driven” by A. castellanii, which would be dependent on amoeba intracellular signal transduction pathways involving Src kinases. In contrast, neuropathogenic E. coli K1 is an invasive strain. Their invasion of A. castellanii is most likely reliant on bacterial invasion, which might explain the involuntary uptake of E. coli K1 by A. castellanii, as observed in the present study. However, this contrasts with results of previous studies [Staphylococcus aureus-invasion of embryonic kidney cells, Shigella invasion of epithelial cells and uropathogenic E. coli-invasion of bladder epithelial cells, which may be due to different host cell types, i.e., phagocytic Acanthamoeba versus non-phagocytic host cells. Future studies are needed to address these aspects in a comparative way. studies ,28,29, wAcanthamoeba are known to secrete large amounts of proteases that are shown to be key virulence factors in keratitis [Acanthamoeba increased the permeability of the blood–brain barrier by over 45% [A. castellanii. The proteases involved were shown to be serine proteases, inhibited by PMSF. This is significant as the inhibition of serine proteases dramatically reduce the ability of A. castellanii to cause inflammation and tissue damage [eratitis ,31 as weeratitis ,13. For over 45% , while ie damage  and possA. castellanii to the host cell is an important step in the pathogenesis of acanthamoebiasis [A. castellanii adhesion to HBMEC, which is not surprising, as Src kinases are regulators of intracellular signalling pathways, and the binding of amoebae to HBMEC is known to be mediated by mannose-binding protein expressed on the surface membranes of Acanthamoeba[A. castellanii and that both properties have been implicated in the pathogenicity of Acanthamoeba, it was anticipated that inhibition of Src kinases would result in reduced A. castellanii-mediated HBMEC cytotoxicity. It was surprising that the inhibition of Src kinases had no significant effect on HBMEC cytotoxicity. However, cytotoxicity is a delayed event and PP2 effects are reversible [A. castellanii with PP2 for 30 min followed by their incubation with HBMEC for 24 h. in the absence of inhibitor, may explain these findings. As PP2 has toxic effects on the host cells, assays could not be performed in the presence of PP2 for longer periods of time. Future studies, using host cells expressing dominant negative forms of Src kinases, should establish their involvement in A. castellanii-mediated HBMEC cytotoxicity.Previous studies have shown that the adhesion of oebiasis ,34. Our nthamoeba,34. Giveversible . The facA. castellanii. Future studies should further explore the precise mechanisms associated with Acanthamoeba pathogenesis, which may help develop preventative and/or therapeutic interventions against acanthamoebiasis.For the first time, the present findings showed that Src kinases are involved in the proliferation, phagocytic properties and proteolytic activities of The authors declare that they have no competing interests.NK conceived the study. RS and MM designed and conducted all experiments under the supervision of NAK. JI performed genome analyses and interpretations. RS, JI and NAK contributed to the writing of the manuscript. All authors approved the final manuscript.


Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function.Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study.2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization.NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for the treatment of cancer and various inflammatory diseases.Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca Sesamum indicum L., an important oilseed crop i mobilization, and finally inhibition of platelet aggregation -5-isoquinolinesulfonamide dihydrochloride (H89), and 1H-, then apyrase (1.0 U/ml), PGE1 (0.5 μM), and heparin (6.4 IU/ml) were added, and the mixture was incubated for 10 min at 37°C. After centrifugation of the suspensions at 500 g for 10 min, the washing procedure was repeated. The washed platelets were finally suspended in Tyrode's solution containing bovine serum albumin (BSA) (3.5 mg/ml) and adjusted to about 4.5 × 108 platelets/ml. The final concentration of Ca2+ in the Tyrode's solution was 1 mM.Human platelet suspensions were prepared as previously described . This st8 platelets/ml) were preincubated with various concentrations of sesamol or inhibitors for 3 min before the addition of collagen (1 μg/ml). The reaction was allowed to proceed for 6 min, and the extent of aggregation was expressed in light-transmission units.A turbidimetric method was applied to measure platelet aggregation , using ag for 10 min. The PRP was incubated with Fura 2-AM (5 μM) for 1 h. Washed platelets (8 × 108 platelets/ml) were then prepared as described above. Finally, the external Ca2+ concentration of the platelet suspensions was adjusted to 1 mM. The rise in the [Ca2+]i was measured using a fluorescence spectrophotometer  with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm i mobilization, and finally inhibition of platelet aggregation. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two secondary messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) i mobilization and platelet aggregation. BAY11-7082, at low concentration of up to 10 μM, not only significantly attenuated [Ca2+]i mobilization, but also inhibited platelet aggregation stimulated by collagen  formation i mobilization, and platelet aggregation , resulting in the production of the second messengers, IP3 and DAG i mobilization, and finally inhibition of platelet aggregation. Therefore, sesamol may represent an increased therapeutic potential to treat such thromboembolic disorders. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our present data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for treating cancer and various inflammatory diseases.In conclusion, the most important findings of this study demonstrate for the first time that the antiplatelet activity of sesamol may involve an increase in cAMP/PKA, followed by inhibition of NF-κB-PLC-PKC signaling events, which leads to inhibition of [CaThe authors declare that they have no competing interests.CCC performed research and wrote the manuscript; WJL, ETO, CWC, and SCL performed the partial experiments and analyzed data; SYH and JRS conceived of the study and designed research. All authors read and approved the final manuscript.


The recent development of mammalian target of rapamycin (mTOR) kinase domain inhibitors and genetic dissection of rapamycin-sensitive and -insensitive mTOR protein complexes (mTORC1 and mTORC2) have revealed that phosphorylation of the mTOR substrate 4E-BP1 on amino acids Thr37 and/or Thr46 represents a rapamycin-insensitive activity of mTORC1. Despite numerous previous reports utilizing serine (Ser)-to-alanine (Ala) and threonine (Thr)-to-Ala phosphorylation site mutants of 4E-BP1 to assess which post-translational modification(s) directly regulate binding to eIF4E, an ambiguous understanding persists. This manuscript demonstrates that the initial, rapamycin-insensitive phosphorylation event at Thr46 is sufficient to prevent eIF4E:4E-BP1 binding. This finding is relevant, particularly as mTOR kinase domain inhibitors continue to be assessed for clinical efficacy, since it clarifies a difference between the action of these second-generation mTOR inhibitors and those of rapamycin analogues. Well-characterizedin vivo substrates of rapamycin-sensitive mTOR activity include the 70 kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic [translation] initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The mTOR-dependent phosphorylation site on S6K1, Thr389, is required for kinase activity, explaining rapamycin’s inhibition of S6K1 activity4. 4E-BP1, on the other hand, is subject to multisite phosphorylation culminating in the release of bound eIF4E, leading to an ambiguous understanding of which phosphorylation site(s) regulate(s) eIF4E binding5.The mammalian target of rapamycin (mTOR) protein is an atypical Ser/Thr protein kinase named for its well-characterized inhibition by the natural product rapamycin. Rapamycin-sensitive orthologues of mTOR exist in eukaryotes from yeast to man and are required for growth and proliferation of perhaps all eukaryotic cells. As such, rapamycin has been classified as an anti-fungal agent and is clinically approved as an immunosuppressant and cancer therapy6. This approach of separating post-translationally modified forms of a protein based on charge and apparent molecular weight has proved to be particularly useful when combined with phosphorylation-specific antibodies7. Due to amino acid sequence similarity, phospho-specific anti-Thr37/46 antibodies did not allow determination of whether the initial phosphorylation event is at Thr37 or Thr46 using this technique, although priming phosphorylation at both of these sites is thought to be required for subsequent phosphorylation at Thr70, followed by phosphorylation at an unidentified site, and finally at Ser658. Given their positions flanking the amino acids responsible for eIF4E binding (amino acid residues 53–59), it is conceivable that Thr46 and Ser65 are responsible for the phosphorylation-mediated modulation of eIF4E binding occurring in response to mTOR activity. Indeed, a significant body of work supports the role of phosphorylation at Thr46 in regulating eIF4E:4E-BP1 binding12. Detailed analyses have led, however, to conflicting results regarding the importance of Ser65 phosphorylation in preventing this protein:protein interaction15.Mammalian 4E-BP1 is subject to an ordered phosphorylation on at least 5 major amino acid residues in response to serum-stimulation, as has been demonstrated by two-dimensional (isoelectric focusing and SDS-PAGE) electrophoresis (2DE)17. This residual rapamycin-insensitive phosphorylation is sensitive to serum starvation, amino acid withdrawal, and non-specific phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase (PIKK) inhibitors19. Furthermore, the use of mTOR kinase domain inhibitors (Torin1 and PP242) in combination with mTOR complex 2 (mTORC2)-deficient cells, has allowed the determination that Thr37/46 phosphorylation represents a rapamycin-insensitive function of mTOR complex 1 (mTORC1)21.While rapamycin is effective in blocking phosphorylation at Thr70 and Ser65, phospho-specific antibodies to Thr37/46 show that at least one of these sites is largely rapamycin-insensitiveIn vivo studies addressing the relative importance of 4E-BP1 phosphorylation sites have been hampered by its ordered phosphorylation, wherein Thr-to-Ala mutation of Thr37 or Thr46 will block subsequent phosphorylation at Thr70 and Ser65. Mounting circumstantial evidence supports the notion that phosphorylation of Thr37/Thr46 alone is the key event regulating eIF4E:4E-BP1 bindingin vivo. Intracellular co-localization of endogenous 4E-BP1 and eIF4E best correlates with dephosphorylation at Thr37/4619. 7-methyl-GTP (cap-column) pull down of eIF4E:4E-BP1 complexes is enhanced by mTOR kinase domain inhibitors more than it is by rapamycin21. Most importantly, however, mTOR active site inhibitors capable of blocking phosphorylation at Thr37/46 (and not rapamycin) induce 4E-BP-dependent phenotypes in cells22.23, a thorough understanding of the importance of rapamycin-insensitive mTORC1 activity is crucial. This manuscript supports the idea that clinically used mTOR kinase domain inhibitors will reduce eIF4E availability much more profoundly than have clinically approved rapamycin analogs.This manuscript describes new data demonstrating that 4E-BP1 phosphorylation at the initial, mTORC1-dependent, rapamycin-insensitive phosphorylation site is alone in regulating eIF4E binding. Furthermore, this work suggests that Thr46, and not Thr37, is this key phosphorylation site. Given the recent push for pharmaceutical development of kinase inhibitors that block both the rapamycin-sensitive and rapamycin-insensitive activities of mTOR24. Far western analyses were performed as follows using a 32P-labelled eIF4E protein25 with an N-terminal substrate peptide for heart muscle kinase (HMK). One hundred units of bovine HMK was suspended in 10 µl of 40 mM DTT and allowed to stand for 10 minutes. Five micrograms of HMK-eIF4E protein was mixed with 3 µl 10X HMK Buffer , 5ul [γ-32P] ATP 3000 Ci/mmol, 1 µl HMK (10U), and water (to 30 µl) and incubated for 45 minutes at 4°C. Probe purification was performed using Pharmacia Nick Column Sephadex G-50 DNA grade. Membranes were subjected to pre-hybridization  for 5 hours at 4°C. Probe hybridization was performed in buffer  with 250,000 cpm/ml of radiolabelled probe for 10 hours at 4°C. Membranes were washed with hybridization buffer 3 times, 15 minutes prior to exposure to film . Following far western analysis, membranes were probed sequentially with antibodies for Phospho-Ser65, Thr70, Thr37/46, and Total 4E-BP1 . Cap column based fractionation of cell lysates was performed as previously described26. HeLa S3 and 293 HEK cells were treated with PP242  or Rapamycin  unless otherwise indicated. Stable HeLa S3 cell lines expressing wild-type and mutant HA-4E-BP1 proteins were generated using previously described mammalian expression constructs8 and G418 selection. Control siRNA and siRNA for mTOR was from Cell Signaling Technology and delivered using Lipofectamine2000 according to manufacturer’s instructions (Invitrogen). All antibodies were from Cell Signaling Technology and were used according to manufacturer’s instructions. Compounds used were rapamycin (LC Labs), PP242 (Intellikine), torin1 (Tocris), PI-103 (EMD), etoposide and nocodazole (Sigma). Standard laboratory practices were used to control bias and unwanted sources of variability in this study. The primary limitation of the datasets presented in this manuscript is that they represent single biological replicates of an experimental procedure.Isoelectric focusing combined with SDS-PAGE based two-dimensional electrophoresis was performed as previously described25. Using this approach, we demonstrate that the treatment of cells with the mTOR kinase inhibitor PP242 results in the following: (i) dramatic increases in the eIF4E-binding competent pool of 4E-BP1, (ii) reduction of 4E-BP1Thr37/46 and Ser65 phosphorylation, and (iii) unaffected binding to bands corresponding in molecular weight to 4E-T and eIF4G  are detected by the total 4E-BP1 antibody, with forms B-F phosphorylated at Thr37 and/or Thr46, D-F phosphorylated at Thr70, and F phosphorylated at Ser65. Far western blot analysis demonstrates that only spot A is competent to bind HMK-eIF4E under control conditions suggesting that the modification responsible for spot B (Thr37 or Thr46) disrupts this interaction. Upon inhibition of mTOR with rapamycin or PP242, the predicted decrease in hyperphosphorylated 4E-BP1 forms is observed with an increase in hypophosphorylated 4E-BP1. While the identity of the phosphorylation event responsible for spot E remains undetermined, these data show that this phosphorylation site is resistant to mTOR inhibition, as rapamycin and PP242-resistant phospho-forms emanating from spot E appear above spots B-D. It is of note that the PP242-induced spot above spot B, which is not phosphorylated at Thr37 or Thr46, represents mono-phosphorylated 4E-BP1 (at the site responsible for spot E) and retains eIF4E-binding ability. The most likely candidates for this site are Thr84, which has shown to be responsible for a similarly slow SDS-PAGE migration11, and Ser101, which has been shown to promote 4E-BP1:Raptor binding28. The notion that Ser101 is responsible for spot E is particularly appealing, as this would provide a sound explanation for the hierarchical ordering of this phosphorylation event prior to Ser65 (spot F). That is, it is reasonable to believe that strong 4E-BP1:Raptor binding is required for complete 4E-BP1 phosphorylation, including at Ser65.To more precisely elucidate the molecular modifications of 4E-BP1 induced by PP242 that induce eIF4E binding, whole cell lysates were prepared from HEK293 cells that were subjected to short (30 min) treatment with PP242 or rapamycin followed by 2DE (isoelectric focusing and SDS-PAGE) and western blot analyses . Under cin vivo using the phospho-4E-BP1 Thr37/46 antibody to determine whether the preclusion of phosphorylation at one site blocks phosphorylation at the other  4E-BP1 exists and is eIF4E binding competent. A similar conclusion was recently reached by another group31. It should be noted that the total 4E-BP1 antibody detects a doublet band in the cap-column bound eIF4E fraction. The upper band of this doublet likely represents 4E-BP1 phosphorylated at the site responsible for the above-described "spot E"; a phospho-form which retains eIF4E-binding ability.This model, wherein 4E-BP1 phosphorylation at the initial phosphorylation site (Thr46) is sufficient to prevent eIF4E binding, is also supported by 7-methyl-GTP (cap-column) pull down data . Here thTo further explore the potential existence of 4E-BP1 phospho-forms failing to adhere to the strict hierarchical phosphorylation pattern Thr37/46->Thr70->spot E->Ser65, untreated and nocodazole-blocked HeLa S3 cell lysates were mixed and subjected to 2D-E. Nocodazole block was again used to induce aberrant phosphorylation patterns of 4E-BP1. As shown in32. Notably, both PP242 and Torin1 were shown to bind multiple PI3K-related kinases (PIKKs), including three key DNA damage-activated kinases ATM, ATR and DNAPK, although cell-based assays failed to show inhibition of these kinases. To assess the specificity of PP242 in our system, we employed a broadly reactive phospho-specific antibody capable of recognizing multiple phospho-Ser/Thr-Gln substrates of ATM, ATR and DNAPK34. Indeed, with this antibody, we observed multiple etoposide-induced bands by western blot analysis  failed to identify a single important phosphorylation site11; or (iii) employedin vitro phosphorylation of Thr46Ala point mutant using a non-physiological kinase12.Although, our conclusion that phosphorylation at Thr46 is the key event regulating 4E-BP1:eIF4E binding has been suggested previously, none of these previous studies unambiguously established that Thr46 alone is the key important site. These studies either: (i) employed35. It is interesting to note that both of these studies evaluate the capacity ofin vitro phosphorylation at Thr46 to disrupt pre-existing 4E-BP1:eIF4E complexes and utilize cap column purification to isolate eIF4E-bound 4E-BP1. This experimental detail is particularly relevant, as it has been shown that the RNA cap can exert an allosteric effect stabilizing 4E-BP1:eIF4E binding36. Taken together, our data and these previous reports could suggest that Thr46 phosphorylation is sufficient to block the initial binding between eIF4E and 4E-BP1 as observed by far western analyses, but that hyperphosphorylation of 4E-BP1, including at Ser65, is required to disrupt existing 4E-BP1:eIF4E complexes.Importantly, equally credible work has reported that phosphorylation on Thr46 is unimportant in the regulation of 4E-BP1:eIF4E bindingThe far western analysis of 2DE separated 4E-BP1 phospho-forms presented inde novo association of eIF4E (4E-BP1 associated or not) with an mRNA 5´ cap analog, therefore any pre-existing complexes must dissociate from cellular mRNA caps prior to isolation. These data do tell us that if there is some pool of 4E-BP1 phosphorylated on Thr46 associated with eIF4E, then this complex is not able to efficiently bind the mRNA cap analog. This assay may also evaluate the sequential binding of eIF4E to the cap followed by 4E-BP1 binding to cap-associated eIF4E and indicate in agreement with the far western data inde novo binding is blocked by Thr46 phosphorylation.The data presented in26. The polyclonal antibody, referred to as 11208 and used in this previous study, however, exhibits selectivity for the non-phosphorylated forms of the protein after nocodazole treatment . A similar faulty antibody-based discrepancy regarding the phosphorylation state of 4E-BP1 in meiotic oocytes was resolved37. While we observed at most 6 different phospho-forms of 4E-BP1, the PhosphoSitePlus database38 indicates that 20 distinct phosphorylation sites have been described in the literature and/or mass spectrometry-based datasets. This discrepancy suggests that many of these 20 phosphorylation events are either mutually exclusive, or occur only rarely, not at all, in response to specific stimuli, or in specific cell types.Finally, the demonstration presented within this manuscript that PP242 exhibits poor specificity for mTOR in a cell-based assay for PIKK off-target effects likely does not change the conclusions of this manuscript; however, it serves as a reminder that any new mTOR inhibitor may have unanticipated effects. While the inhibition of mTORC2 by mTOR kinase domain inhibitors is expected to have profound effects, the evidence presented in this manuscript suggests that the rapamycin-insensitive activity of mTORC1 towards 4E-BP1 will also be quite important as the clinical safety and efficacy of mTOR kinase domain inhibitors is assessed. This submission is quite nice for experts, but not particularly well-written for those outside of the field of 4E-BP1 phosphorylation, who will have difficulty reading it ; hence my suggested clarifications below. The article is important as it demonstrates that the use of mTOR-kinase domain inhibitors would be more efficacious than the use of rapamycin analogs as therapeutics.The work clarifies that phosphorylation of 4E-BP1 at Ser65 by mTOR is not required to inhibit 4E-BP1 binding to eIF4E and that rapamycin-insensitive mTOR-mediated phosphorylation at 4E-BP1 Thr46 is sufficient to inhibit 4E-BP1 binding to eIF4E. Phosphorylation at 4E-BP1 Thr37 is also shown to be rapamycin-insensitive. Notably, evidence that phosphorylation at Thr 37 or Thr 46 appears to be required for phosphorylation at Thr 70 and Ser 65 supported the notion that the earlier phosphorylation events were key to promoting translation.Improvement Suggestions:Page 2, first paragraph, last sentence. The meaning of “4E-BP-dependent phenotypes” is not particularly clear.Page 2, line four of Materials and Methods. Shouldn’t “32″ be superscript?Page 2, last paragraph. The statement “Here, six differentially phosphorylated forms (A-F) …” is confusing. Is form A really phosphorylated? Earlier in the manuscript, the authors mentioned 5 major phosphorylation sites.In Figure 1, the assay used in this Figure requires much better description in the text. Part A of the legend requires specification of the experimental approach. Also, the title of Figure 1 could be more useful if it were informative/conclusive rather than descriptive.Page 3, right column, second paragraph. The sentence “While endogenous… , suggesting that Thr46 phosphorylation is required for subsequent The37 phosphorylation under normal growth conditions.” is misleading since 4E-BP1 mutant (Thr46Ala) is clearly phosphorylated at Thr37 in Fig. 2A middle panel. With the data presented here the reader is left wondering whether the differences in detecting HA-4E-BP1 (T37A) or HA-4E-BP1 (T46A) might be simply due to different affinities of the (The37/46) antibody used.What is the difference between the middle and the lower panel? Are the antibodies used to probe the middle and the lower panel (e.g. phosho-HA-4E-BP1 (Thr37/46) vs. phosho-4E-BP1 (Thr37/46)) different or the same? When using cell lysates of stable cell lines and a phospho-4E-BP1 (Thr37/46) antibody, shouldn’t be HA 4E-BP1 wildtype and mutant forms be detectable in the lower panel as well?Page 3, last paragraph, line 8. “modulate” would be clearer if described as the specific modulations that occur – in the literature a state of 4E-BP1 hypophosphorylation. The author understands that the changes are complicated and clarified from these authors’ perspective in Figure 3 and in the Discussion. However, possibly, the reader could be better informed at this point of the manuscript.Page 4, second paragraph, right-hand column. Please clarify the effects and usefulness of etoposide and the meaning of VCP and, in the legend, VCP/Chk2.In Figure 2B, the authors should provide a control western blot of an unrelated protein which does not belong to the eIF4E-associated proteins under normal conditions to show the unspecific background of this assay.It is not clear to this reader why mono-phosphorylated 4E-BP1 (at position Thr70) is detectable in the non-Nocadazole treated cell samples in Fig. 2B (lane 3 and 5) when in Fig.3 they show that mono-phosphorylation of 4E-BP1 at position Thr70 is triggered by a Nocodazol-induced block of Hela cells but not detectable under normal conditions.In Figure 3, why did the authors mix Control and Nocodazole-blocked Hela cell lysates instead of showing them side-by-side? Also, why are there additional spots in the interpretation panels for Phos-Thr70 and Phos-Thr37/46 that have no counterpart in the respective western blot panels? This figure is already difficult, so the authors should be careful with the overlays.In Figure 4, all results in this Figure are not adequately described. For example, the authors need to explain the analysis of ubiquitin and its significance to their studies.The sentence “ …rapamycin-insensitive phosphorylation site is alone in regulating eIF4E binding.” should read something like “ …rapamycin-insensitive phosphorylation site is alone responsible in regulating eIF4E binding”.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Through binding to eIF4E, the 4EBP proteins  interfere with formation of the eIF4F cap-binding complex. A key function of the mTOR serine-threonine kinase is to phosphorylate 4EBPs on multiple sites, which blocks their ability to bind to eIF4E thus potentiating cap-dependent translation.Recently it has become clear that ATP-competitive mTOR kinase inhibitors are more effective than the allosteric compound rapamycin at blocking mTOR-dependent 4EBP phosphorylation, particularly on sites T37 and T46. This greater effect of mTOR kinase inhibitors on 4EBP phosphorylation correlates with increased anti-proliferative and pro-apoptotic effects. The purpose of the current study is to determine the order of phosphorylation of sites T37 and T46, and which site is most important for regulating binding of 4EBP1 to eIF4E. This is a fairly narrow question to address but the authors argue that it is important for understanding drug mechanism and that previous approaches have not addressed this question adequately.Overall the data support the conclusion that T46 phosphorylation is the initial, rapamycin-insensitive event that blocks 4EBP1-eIF4E binding. The main approach used is two-dimensional electrophoresis (IEF followed by SDS-PAGE), then far-western blotting with labeled eIF4E to determine which spots are capable of eIF4E binding. Figure 1A provides a 1-D blot that shows convincingly that the far-western technique can reveal increased binding of eIF4E to 4EBP1 in cells treated with the mTOR kinase inhibitor PP242. Figure 1B shows that 4EBP1 phosphorylation and release from eIF4E are induced by serum in a mTOR-dependent manner. Figure 1C presents the 2D data illustrating hierarchical phosphorylation and distinct effects of rapamycin and PP242. The data support the conclusion that only a small subset of 4EBP forms are capable of binding eIF4E, that these forms lack T37/46 phosphorylation, and that PP242 increases this pool to a greater extent than rapamycin. To distinguish the roles of T37 and T46 they express HA-tagged 4EBP1 and alanine substitution mutants in HeLa cells. The data in Figure 2A show that T46A mutation, but not T37A mutation, blocks detection by the phospho-T37/46 antibody. This suggests that T46 phosphorylation precedes and is required for T37 phosphorylation. A cap binding assay in Figure 2B supports the conclusion that T37/46-phosphorylated 4EBP1 cannot bind eIF4E in the cap-binding complex, but this is not novel and the technique does not add further evidence for initial T46 phosphorylation.The rest of the data seem added on and superfluous to the main message of the paper. The experiment in Figure 3 shows 2D far western blotting data on lysates of nocodazole-treated cells, which artificially increases the diversity of 4EBP1 spots. As the authors point out, these might not exist under physiological conditions so the relevance is unclear. Figure 4 addresses an entirely different problem, namely the lack of selectivity of the PP242 compound. While it is of some interest that PP242 differs from other compounds of this class in its apparent inhibition of DNA-PK and related kinases, this observation is too peripheral to the main study and should be developed further before publication.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in  Phosphorylation on a particular Thr residue at their activation loop is one of the most common mechanisms that activate protein kinases In this report we show that the yeast MAPK Hog1 is also regulated by induction of its autophosphorylation activity. Furthermore, we mapped a novel domain in Hog1 that inhibits this activity and an adjacent domain that is critical for induction of autophosphorylation.Hog1 is the yeast ortholog of the mammalian MAPKs JNK and p38. It was discovered as an osmostress-responsive kinase, but is known now to respond to various cues pbs2Δ cells Hog1 is phosphorylated and activated to some degree in response to high osmotic pressure (0.8 M or 1 M NaCl). We report that this Pbs2-independent, osmostress-dependent Hog1 activation is achieved primarily via autophosphorylation. We mapped a C-terminal domain in Hog1 that restricts autoactivation. Removal of this domain increases Pbs2-independent Hog1 activity to levels that allow pbs2Δ cells to proliferate to some degree under osmotic pressure. We further mapped a small region (seven amino acids long) in Hog1’s C-terminal domain to be important for it’s autophosphorylation activity. This region partially overlaps with the Pbs2-interacting site, suggesting that Pbs-dependent and Pbs2-independent mechanisms of Hog1 activation use a similar domain. Finally, we found this region to be functionally conserved in the human p38α.Pbs2 is considered the only MAP2K for Hog1. On its absence therefore, Hog1 should not be phosphorylated and activated and should not support proliferation under high osmostress S. cerevisiae strains used were: JBY13 , a hog1Δpbs2Δ strain 2SO4, 0.5% ammonium sulfate, 2% glucose and 40 mg/liter of the required nutrients. To induce osmotic stress YPD supplemented with 0.8 M NaCl or 1 M NaCl was used.The 600∼0.4). Five serial dilutions were created  and 5 µl from each dilution were plated on YPD plates supplemented with 0.8 M or 1 M NaCl. Plates were grown at 300C for 5 days.Yeast cultures were grown in liquid medium, [YNB(-URA)], to logarithmic phase  using the primers 5′-gatacctggcgtgttatgatgcataagattggtggcagtgat-3′ and 5′-atcactgccaccaatcttatgcatcataacacgccaggtatc-3′. Appropriate removal of the fragment was verified by sequencing. pcDNA-p38α and pBabae-MKK6EE were described previously All 600∼0.5), precipitated by centrifugation  and re-suspended in YPD medium with or without NaCl. At each relevant time point a sample of 20 ml was collected and precipitated. The yeast pellet was washed with 10 ml of 20%TCA and precipitated again by centrifugation . The washed pellet was re-suspended in 200 µl of 20%TCA and 600 mg of glass beads were added. Cells were broken by vortexing for 8′ and the supernatants were transferred to a clean tube. The beads that were left in the tube were washed twice with 200 µl of 5%TCA, and the supernatants were combined to the same tube. The proteins were precipitated by centrifugation  and the pellet was re-suspended in 200 µl of Laemmli buffer and 100 µl of 1 M Tris base and boiled for 3′.Yeast cultures were grown in liquid medium YNB(-URA) to logarithmic phase , anti HA antibodies , anti-MAPKAPK2 , anti-phospho MAPKAPK2  and anti-flag .2. Cells were transfected with pcDNA and pBabe plasmids containing the cDNA of p38α and MKK6EE respectively by the calcium phosphate method as described HEK293T cells were grown in Dulbeco’s MEM medium supplemented with 10% fetal bovine serum and antibiotics. Cells were grown at 37°C under 5% COpbs2Δ cells can proliferate under osmostress, if they express intrinsically active Hog1 molecules such as Hog1D170A, Hog1F318S or Hog1F318Lpbs2Δ cells   . This ex1 M NaCl . Thus, ipbs2Δ cells occurs on the TGY motif. We therefore expressed Hog1T174A, Hog1Y176F, or Hog1T174A+Y176F in pbs2Δ cells. These proteins were not recognized by the anti-phspho-p38 antibodies confirming that Thr174 and Tyr176 are the phosphoacceptors in pbs2Δ cells (data not shown).Although phosphorylation was monitored with site-specific antibodies , we wished to further confirm that Hog1 phosphorylation in pbs2Δ cells could be explained by two possible mechanisms. One is phosphorylation by another MAP2K that partially takes over Pbs2 activity and the other is autophosphorylation. To reveal the mechanism, a kinase-dead Hog1  was expressed in pbs2Δ cells. If Hog1’s activation in these cells is achieved by autophosphorylation, the kinase-dead mutation should abolish its phosphorylation. Indeed, this protein was not phosphorylated under osmotic stress conditions in pbs2Δ cells  that are more similar to p38α  that was mapped by pull down assays to W320-D350 hog1Δ cells the truncated proteins were spontaneously phosphorylated to some degree  as a domain that possesses some inhibitory properties on the protein’s Pbs2-independent activation. To assess if we identified the entire inhibitory domain or perhaps further deletions would increase spontaneous phosphorylation and improve growth of hog1Δ strain revealed that all proteins, with the exception of Hog1V313, were able to support proliferation of these cells under high osmotic pressure  an inhibitory domain of Hog1’s Pbs2-independent activation, includes the region from F343 down to the end of the protein (Q435). 2) A domain essential for autoactivation (Pbs2-independent) and also important for Pbs2-dependent phosphorylation (Y337–F343). 3) A domain (D315–Y337) that functions in the Pbs2-dependent phosphorylation of the protein, but is not essential for the biological activity of the Pbs2-Hog1 system . Only proteins shorter than Hog1(Y337–F343)Δ). When expressed in hog1Δ cells, the Hog1(Y337–F343)Δ protein was biologically active just as Hog1WT, allowing yeast growth under osmotic stress Δ’S phosphorylation was barely detectable and did not allow any proliferation under osmostress . Accordingly, all proteins that allowed proliferation of pbs2Δ cells under osmostress,  and all were phosphorylated in response to osmostress in hog1Δpbs2Δ cells at levels similar to those of Hog1WT Δ maintained autophosphorylation capability. p38αWT, which is constitutively autophosphorylated in yeast (Y342–F348)Δ is not phosphorylated in yeast, suggesting that it lost its autophosphorylation ability.The region identified to be essential for Hog1’s autoactivation, Y337–F343, is conserved in its mammalian ortholog p38α  as well D176A+F327S protein. The p38αD176A+F327S mutant manifests constitutive autophosphorylation and catalysis in vitro and in vivo Δ, p38αD176A+F327S and p38αD176A+F327S(Y342–F348)Δ when transiently expressed in HEK293 cells with or without co-expression of the active form of MKK6 (MKK6EE) which is a MAP2K of p38α. The results showed that in the absence of MKK6EE p38αD176A+F327S is phosphorylated and is capable of spontaneously phosphorylating the p38α substrate MAPKAPK2 (MK2) Δ is not phosphorylated at all (lane 4). Both p38α(Y342–F348)Δ and p38αD176A+F327S(Y342–F348)Δ could be phosphorylated and activated by the active MKK6, but to a lower level than p38αWT  than we did (1 M NaCl). We, too, did not observe Hog1 phosphorylation in pbs2Δ cells exposed to a weaker pressure. We think that autophosphorylation of Hog1 might be biologically relevant because when sufficiently elevated, either by removing the C-terminal inhibitory region, or by insertion of activating mutations, it could relieve osmostress sensitivity of pbs2Δ cells. Our deletion analysis pointed at a 7 amino acids fragment in the C-terminus of Hog1 . This notion is supported by the fact that some of the intrinsically active Hog1 mutants we described previously, such as Hog1Y68H and Hog1A314T, manifest very low phosphorylation levels and catalytic activity in pbs2Δ cells, but are still capable of supporting growth of these cells under osmostress Induction of Hog1WT undergoes some constitutive spontaneous phosphorylation reactions that are futile because they are instantly eliminated via a negative feedback machinery that probably activates phosphatases PTC1 and PTP2 is lethal unless the HOG1 pathway is inactivated KSS1 and FUS3 and in tyrosine kinases too We do not know how the C-terminal inhibitory domain, Q435–L341, controls autophosphorylation. Its sequence does not contain any known binding site or a motif that can disclose its mechanism of action. Also, the sequence is specific to Hog1 and does not appear in any other proteins in the database. This domain may interfere with the ability of the nearby domain to impose autophosphorylation. It could also be that this domain is important for Hog1’s association with phosphatases. Several lines of evidence suggest that Hog1What could be the role, if any, of the autophosphorylation process in the wild type cell? Several explanations may be suggested. For example, to maintain, as a sort of cellular memory, low levels of active Hog1 after stress is relieved and Pbs2 activity is reduced. Autophosphorylation may also function to activate Hog1 in some sub-cellular compartments in which Pbs2 does not exist. Finally, it could be that in the natural activation process Pbs2-depenednet phosphorylation is multiplied by autophosphorylation.Many kinases have the ability of autophosphorylation which plays an important part in their activation Thus, although the biological and physiological role of this type of activation is not clear yet, activation of MAPKs by autophosphorylation seems to be an evolutionarily conserved general phenomenon.


Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock.Protein Y (PY) is an  Escherichia coli cells transiently stop growing for a period which can last one to several hours and enter an acclimation phase during which bulk protein synthesis is drastically repressed while a set of cold-shock genes is selectively and transiently expressed , cis-acting elements present in the cold-shock transcripts which render these mRNAs suitable for the translation in the cold and to trans-acting factors  a drastic stabilization of the transcripts , known also as RaiA or YfiA -pLYS was used for protein over-expression and purification  medium and in M9 minimal medium  and by assaying colony-forming units on LB-plates.Growth curves of E. coli MRE600 and MRE600ΔyfiA grown at 37°C till OD600 = 0.6 were transferred to 10°C. Aliquots of both cultures were taken immediately before or after 60 min of cold shock. The cells were then subjected to a freeze–thaw and lysozyme procedure ; 0.05% pararosa aniline). Two-dimensional (2D) gel electrophoresis was performed with a 4–5 pH gradient for the first dimension and a 15% sodium dodecyl sulfate poly acrylamide gel elctrophoresis (SDS-PAGE) as the second dimension, essentially as described  and then chased for 5 min by adding nonradioactive methionine and cysteine to a final concentration of 0.2 mol/L. Two control samples were also pulse-labeled in the same way for 10 min at 37°C before the cold shock. After centrifugation, each cell pellet was resuspended in 200 μL of phosphate buffered saline buffer containing 1 mmol/L DTT and 0.2 mmol/L phenylmethanesulfonylfluoride (PMSF). The cells were then lysed by sonication and the resulting cell extracts were cleared by centrifugation. The total cell extracts thus obtained, normalized for the A600 value of the corresponding cell aliquot, were subjected to SDS-PAGE at various concentration . The radioactivity incorporated into the proteins was determined with a GS 250 Molecular Imager .Pulse-labeling experiments were performed on cultures grown in M9 minimal medium /pLYS was transformed by the resulting vector pETM11-PY. The pETM11-PY construct was confirmed by DNA sequencing.The coding region of _yfiA mutant was produced using the QuikChange Site-Directed Mutagenesis Kit  from Stratagene and the pETM11-pY plasmid as DNA template, with the following mutagenic primers: 5′-CCACTGGCAACCAGACAGCCGTTAGGTGTATTG-3′ and 5′-CCAAGGCCGTACTCCAACTGTAATCAAAAAAGCAA-3′. The resulting construct pETM11-Val60Cys_PY was sequenced to confirm the mutation and transferred in E. coli BL21 (DE3)-pLYS by electroporation.The Val60CysE. coli BL21 (DE3)-pLYS cells harboring pETM11-PY or pETM11-Val60Cys_PY, grown in LB medium at 37°C to OD600 = 0.4. After transferring the cultures to 20°C for 12 h, the cells were harvested by centrifugation and the pellets were resuspended in Buffer A  and stored at −80°C. After thawing, the cells were diluted in an equal volume of Buffer B , lysed by sonication, and the resulting cell extracts were cleared by centrifugation. The cell extracts were subsequently loaded on a nickel-nitrilotriacetic acid (Ni-NTA) chromatographic column equilibrated in Buffer C . The column, initially washed with the same buffer, was subsequently washed with Buffer D . Protein PY was eluted from the column with Buffer E , and the fractions containing protein PY were pooled and dialyzed against Buffer F . To remove the his-Tag sequence from PY protein, 8.4 mg of purified PY was then incubated for 4 h with 0.5 mg of the His-Tag TEV protease  was induced by the addition of 1 mmol/L isopropyl-beta-D-1-thiogalactopyranoside to E. coli JM109. Namely, these plasmids were pUTcspA , pUT7cspB, puT7cspE, pUT7cspG, pUT7cspI (this study), pTZ19hupA, pTZ19hupB  or pUT7 -1-piperazineethanesulfonic acid-KOH (pH 7.5), 10 mmol/L MgCl2, 50 mmol/L KCl. In vitro translation tests were carried out essentially as described  contained 20 mmol/L tris-HCl, pH 7.7, 80 mmol/L NHThe PYVal60Cys mutant was labeled with the fluorescent dye Alexa555 essentially as described in Milon et al. . The exc4Cl in a Stopped-flow apparatus Kintek SF-2004  by mixing at 15°C equal volumes (20 μL) of reactants present in syringe A or syringe B. The final concentration of the molecules used in the various experiments is indicated in the figure legends. Curves were fitted with second-order equation using GraphPad Prism software. In a single experiment, 1000 data points were acquired, usually in logarithmic sampling mode. Each trace is the average of at least 10 different shots. The fluorescence and light scattering values detected at time = 0 were subtracted from each of the 1000 point which constitute each trace.All experiments were carried out in 10 mmol/L tris-HCl, pH 7.7, 7 mmol/L Mg acetate, 60 mmol/L NHBinding of PY to naked 30S, 50S, and 70S ribosomes was followed by fluorescent change in PY labeled with Alexa555. Syringe A contained PY_Alexa555, while syringe B contained the 30S, the 50S, or the 70S tight coupled ribosomes. For the release assays, 30S-PY_Alexa555 complexes were rapidly diluted with an equal volume of reaction buffer. For both types of experiments, fluorescence excitation was performed at 552 nm and output monitored using a KV590 nm filter.Inhibition of 30S initiation complex formation by PY was followed by fluorescence resonance energy transfer (FRET) between fMet-tRNA_fluo (donor) and IF3 labeled with ALEXA 555 (acceptor), prepared as described  and 50S ribosomal subunits (syringe B) were allowed to mix in 10 mmol/L tris-HCl pH 7.7, 5 mmol/L Mg acetate, 60 mmol/L NHYfiA deletion mutant was generated in E. coli strain MRE600 as described in Experimental Procedures. The deletion of the gene encoding protein PY from the E. coli chromosome was assessed by PCR, as shown in A wt and ΔyfiA strains at 37°C in LB .The cold-shock analysis of  be seen B that thE. coli MRE600 wt and MRE600 ΔyfiA, these strains were grown at 37°C in M9 minimal medium and shifted to 10°C when OD600 = 0.8. At the indicated times , cold-tolerant (ct), and non–cold-shock (non-cs) mRNAs. Among the first class, we selected trans-acting factors, which are expressed only at a later stage of cold adaptation  or subjected to 120 min of cold shock (csS30). These experiments were carried out: (i) at 37°C with control S30 to reproduce the milieu existing when cells grow under physiological conditions; (ii) at 15°C with control S30 to reproduce the conditions existing at the onset of the cold shock when translation occurs in the absence of the cs 027-C mRNA and cspG mRNA (45% inhibition), whereas translation of the other mRNAs was either marginally affected (20–30% inhibition) or affected only at the highest concentration of PY . The inhibition observed in the presence of the csS30 extract seems overall more pronounced (027-C mRNA (80% inhibition) and cspA mRNA, which was repressed only at the highest PY concentration. The fact that inhibition was found to be somewhat stronger with the cold-shock cellular extract could be explained either by the presence of endogenous PY associated with the cs ribosomes or by the presence of other unidentified cs factors capable of reinforcing the inhibitory activity of this protein.The results show that at 15°C, PY inhibited to different extents the translation of all mRNAs examined, with both control S30 A and csSonounced B than thTaken together, these results demonstrate that PY reduces translation in a temperature- and mRNA-dependent manner. This activity is rather modest under the conditions tested, in agreement with the in vivo data which do not support a central regulative role of protein PY during cold stress.35S]Met-tRNA, and 50S subunits in the presence of increasing concentrations of PY and the amount of f[35S]Met-tRNA bound in the 70S IC was determined by nitrocellulose filtration.Previously published data suggest that PY may inhibit two steps of the translation process, namely initiation  and, in addition, indicate that this inhibition takes place mainly at the level of initiation complex formation. In fact, in most of the cases the curves describing the effect of PY on translation (closed symbol) and 70S IC formation (open symbol) are perfectly  or almost perfectly (hupA and cspE mRNAs) superimposable. On the other hand, the behavior of cspE, cspG, hns, and cspI mRNAs is different from that of the other transcripts; in these cases, it can be hypothesized that further inhibition by PY occurs at the level of elongation. It remains to be elucidated why and how PY could affect the translation elongation step of only some and not other mRNAs.The effect of PY on both the entire translational process and the single initiation step is shown in To understand the mechanism of action of PY, binding of this protein to the ribosomal subunits and to the 70S ribosome was studied at 15°C by monitoring the fluorescence change of an engineered PY in which the native Valine in position 60 was replaced by a Cysteine  to allow the labeling of the protein with fluorophore Alexa555 (PY_Alexa555). Control experiments showed that Val60Cys_PY has an activity comparable to the native PY (data not shown) and that an increase in fluorescence can be observed only when the PY_Alexa555 is mixed with the ribosome A.kapp1 × x) + A2 × exp (−kapp2 × x), a two-step binding of PY to all macromolecules tested may be hypothesized. In the above mentioned equation, kapp1 and kapp2 refer to the apparent rate constants of the first and second phase and A1 and A2 to the respective amplitudes.As the experimental curves obtained by mixing PY with the 30S subunit, the 50S or the 70S ribosome A can be kon) and dissociation (koff) rate constants of the binding reactions, the values of kapp1 and kapp2 were determined as a function of increasing 30S and 50S subunit concentrations (kon1 = 12.1 ± 2.1 μmol/L per sec and koff1 = 4.0 ± 2.1 sec−1 for the binding to the 30S subunits (kon1 = 1.2 ± 0.6 μmol/L per sec and koff1 = 6.5 ± 0.8 sec−1 for the binding to the 50S subunits (kapp2 (≅0.3 s−1 for both 30S and 50S) were shown to be concentration independent  can be calculated, which in turn can be used to derive the dissociation constant of the binding reactions (kd = kd1 × kd2), corresponding to 83 nmol/L for 30S-PY and 1.26 μmol/L for 50S-PY.To calculate the  complex . As kappIn conclusion, these experiments show that PY binds rapidly and tightly to the 30S ribosomal subunits and very slowly and weakly to the 50S subunits. As for the binding to the 70S ribosome, it probably occurs with an intermediate affinity.Finally, to determine whether the PY binding to the 30S subunits is reflected in a change in the association rate of the small and large subunits, formation of idle 70S ribosomes was studied following the increase in light scattering at 15°C. The result of the experiment clearly shows that PY accelerates the kinetics of 70S ribosome formation about two times E.cspG mRNA and 027-C mRNA. Increasing amounts of protein PY were added to the reaction mixtures either simultaneously with IF1, IF2, IF3, mRNA, f[35S]Met-tRNA, 30S and 50S subunits or after the same components were preincubated for 15 min at 15°C to preform 70S initiation complexes. Our results  when formed in the presence of PY (closed circles), whereas it was strongly inhibited (>80%) when PY was mixed with the 30S and 50S subunits before the addition of IFs and mRNA (open circles). The same experiment was repeated also in the absence of the 50S subunits  was observed when protein PY was prebound to the 30S subunits, before the other components were added. On the other hand, if PY was added to the reaction after preincubating for 15 min cspA mRNA, 30S subunit, IFs, and S100 postribosomal supernatant, inhibition was reduced (20% inhibition at 15°C and <10% inhibition at 37°C). Note that the stronger inhibition at 15°C than at 37°C confirms that this protein is able to affect translation mainly at low temperature.To confirm the result of these experiments, we performed in vitro translational tests B at 15°CThe scarce inhibition observed when PY is not prebound to the 30S subunits probably implies that this protein does not compete efficiently with the other ligands  for the binding to the 30S. To verify our supposition, the fluorescence change in PY_Alexa555 was monitored at 15°C in the presence of various combinations of ribosomal ligands prebound to the 30S subunit C. All tiE. coli strain (MRE600) and its isogenic strain lacking the gene encoding for protein PY (yfiA) (MRE600 ΔyfiA), we demonstrate that neither the recovery from the cold stress , regardless the partial or full activity of the ribosome degradation pathway. PY would cause also the partial inhibition of the translation of some mRNAs, while those amenable to initiate efficiently translation in the cold by rapidly forming 30S initiation complexes, such as cspA mRNA, would not be affected by the PY activity.Taken together our findings suggest a new model which can explain the role of PY during cold shock. Upon lowering the temperature, protein synthesis diminishes possibly because of unfavorable mRNA secondary structures capable of reducing the rate of the initiation complex formation. PY would bind to a fraction of the free 30S subunits derived from this cold-induced polysome dissociation (Uchida et al.