The ‘phosphate-binding tag’ (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a ‘closed system’ since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin  induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional ‘loading’ or ‘reference’ standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here. The cellular responses mediated by protein phosphorylation are vast in number and function 2+-phos-tag SDS-PAGE permits the study of the effects of experimental treatments on the target protein across its various phospho-states by probing replica membrane with an anti-total-target protein Ab and enables the study of protein phosphorylation in the absence of phospho-protein-specific Abs. Where phospho-protein-specific Abs are available, the technique yields information regarding the distribution of specific phosphoisotypes  across various phospho-states . As such, this technique permits identification of the phospho-states corresponding to specific phosphoisotypes. A previous report using Mn2+-phos-tag SDS-PAGE to assess phosphorylation of myosin regulatory light chain (RLC) demonstrated the enhanced sensitivity of the technique In traditional phospho-protein analyses by WB, the signal derived from a phospho-specific antibody (Ab) toward the target protein is normalized to a reference protein, or to the ‘total’ (‘bulk’) target protein using an Ab that does not discriminate between phosphorylated and non-phosphorylated forms. This type of analysis is normally limited to measuring changes at a single phospho-site per assay. In contrast, MnAs an exemplary model, we have measured the phospho-state distribution of RLC in human uterine myocyte lysates under various experimental conditions. The contractility of uterine and other smooth muscle (SM) beds is dependent on the state of phosphorylation of RLC. In particular, phosphorylation of RLC at S19 triggers cell shortening and force production at the tissue level A potential complication in this phospho-RLC analysis is that a single phospho-state could contain more than one phosphoisotype. Similarly, one phospho-site might appear in more than one phospho-state 2+-phos-tag SDS-PAGE these three Abs yield distinct banding patterns. The CtRLC Ab  of the others, and vice versa. Use of this ‘closed system’ concept has several advantages that will be discussed in a later section that will address its validation.To demonstrate quantifiable changes in the phospho-state distribution of RLC, we treated primary cultures of uterine myocytes with pharmacological agents likely to alter to alter phosphorylation of RLC. RhoA and rho-kinase (ROK) are known to regulate uterine contractility Calp has been shown to specifically activate rhoA in fibroblasts cultures, possibly by influencing a protein tyrosine-phosphatase upstream of rhoA T]/, etc.), and averaging these proportions for n = 5–8 samples. This ‘sum method’ amounts to performing an ‘in-lane’ normalization, in place of that commonly achieved by using a loading control. We propose that this ‘in-lane normalization’ method has several advantages that are discussed in a subsequent section. Note that the WBs have been intensified for all figures so that all three bands are visible, where possible. This is achieved by restricting the upper and lower boundaries of pixel intensity that are shown by the quantification software. The quantitative estimates of band intensity are completely independent of these manipulations. The blots presented here did not exhibit pixel saturation, and therefore have not exceeded the upper limit of the scanner in detecting higher protein loads.This analysis has two primary goals: 1) isolating the effect of the treatment of interest from any vehicle-induced change in the phospho-state distribution, and 2) facilitating comparisons across treatments by expressing them relative to a common reference point (untreated distribution). T, 36±2% by 1pRLCT, and 16±3% by 2pRLCT. Next, we corrected for the effects of the vehicles by subtracting the vehicle-induced changes from those of the corresponding treatments. The net treatment-induced changes are presented in T to 86±2% from 47±4% in untreated samples, whereas 1pRLCT and 2pRLCT fell to 7±2% and 6±2% respectively. Calp (n = 8) treatment causes a specific increase in 2pRLCT from 16±3% to 38±8%, in agreement with the increase in p18p19RLC shown in T is accompanied by a corresponding reduction in 0pRLCT from 47±4% to 29±6%, but no change in 1pRLCT (from 36±2% to 34±4%). These finding suggest that activation of rhoA, and presumably ROK, is associated with a specific increase in the intracellular concentrations of diphosphorylated RLC. OT appears to enhance both 1pRLCT and 2pRLCT, perhaps favouring the former .T state appears to be strongly induced by the vehicle, and most likely results from the mechanical stimulation of cells by pipetting the treatment solution. The short duration of treatment (20 sec) prevents this stimulatory effect from returning to basal levels. Importantly, the OT vehicle does not reduce 0pRLCT as OT does  in-lane normalized to correct for variations in protein loading and signal intensity, 2) vehicle normalized to ascertain that the measured effect can be attributed to the drug or treatment of interest, and 3) normalized to the untreated distribution to permit comparisons across treatments.2+-phos-tag acrylamide gels for several reasons. First, the physical separation of phospho-states along the vertical dimension of the gels increases the likelihood that the loading control protein overlaps with a band corresponding to the protein of interest. Second, if it is phosphorylated, the loading control protein might dissociate into two or more bands and might complicate identification and quantification of loading control and the protein of interest on the same blot. Despite these potential problems, to validate the ‘closed system’ concept we performed a traditional loading control quantification of the phospho-states detected by the CtRLC Ab using SM α-actin as our in-lane reference.The method for data normalization of a closed system described above has advantages over normalization to an in-lane loading control. Specifically, the use of a traditional loading control might not be feasible in Mn2+-phos-tag SDS-PAGE separation without any evidence of phosphorylation under our experimental conditions. Though we were fortunate to have chosen α-actin, finding a loading control protein that does not suffer from at lease one of the above-mentioned problems might prove difficult. If a single-channel detection method is used, WB quantification might be unfeasible. Where dual-channel detection is used for segregating the loading control and target protein signals, the primary-Ab-secondary-Ab-fluorophore combinations must be of high specificity. If used appropriately, the ‘closed system’ method for data quantification proposed here circumvents all of these difficulties by eliminating the need for measuring a loading control protein.T, 1pRLCT, and 2pRLCT in the vehicle and treatment lanes were normalized individually to the in-lane α-actin signal, without expressing them as the proportions of the sum-total of signals as was performed to obtain T relative to 1pRLCT and 2pRLCT. RhoA activation with Calp causes a large increase in 2pRLCT. OT treatment causes an enhancement of both 1pRLCT and 2pRLCT that is reflected as a dramatic reduction in 0pRLCT.For this validation, the signals for 0pRLCT, 1pRLCT, and 2pRLCT in the treatment groups relative to their proportions in the vehicle groups. No significant differences between the ‘closed system’ (‘sum method’) and the traditional loading control methods were observed. Both methods show that g-H treatment causes an increase in 0pRLCT , and a reduction in 1pRLCT  and 2pRLCT . Calp causes increased 2pRLCT . OT causes virtually complete phosphorylation of the pool of RLC, as evidenced by near abolition of 0pRLCT  and an increase in 1pRLCT  and 2pRLCT . The remarkable agreement between these α-actin normalized data and those in To provide a direct quantitative comparison between the 2+-phos-tag separation. In preliminary experiments, we validated the specificity of two anti-phospho-S19-RLC Abs by WB after Mn2+-phos-tag SDS-PAGE separation , etc.). For numerical data, see One important distinction between the data provided by the p19RLC and the CtRLC Abs is that the p19RLC data does not represent a closed system, since it is unable to provide information about 0pRLC or phospho-states that do not include phospho-S19. Therefore, the use of p19RLC Ab in this setting cannot assess quantitative changes in phospho-states, but can provide information about whether the proportions of phosphoisotypes (containing phospho-S19-RLC) within each phospho-state have changed. In 19 and 2pRLC19 are 60±3% and 40±3%, respectively. g-H treatment causes a shift in these proportions to 91±7% and 9±4%, suggesting that ROK inhibition causes specific loss of 2pRLC containing phospho-S19-RLC. However, the data provided earlier in 19 and 2pRLC19 to 43±6% and 57±5%, respectively, reflecting the specific enhancement in 2pRLC seen in 19 and 2pRLC19 to a similar extent, even if the total cellular content of 1pRLC is larger, as evidenced by the data in In the untreated group, the relative proportions of 1pRLCT and 2pRLCT relative to OT alone, and reduced 2pRLCT synthesis relative to Calp alone. Interestingly, ROK inhibition with g-H appears to completely abolish the ability of either stimulant to induce 2pRLCT. In the case of OT+g-H, the reduced 2pRLCT is accompanied by an accumulation of 1pRLCT relative to OT alone. In contrast, the effect of g-H on Calp has little to no effect on 1pRLCT, but results in an accumulation of 0pRLCT relative to Calp alone. These data suggest that the mechanisms utilized by OT and Calp to enhance phospho-RLC concentrations are different. However, both stimulants utilize ROK to induce 2pRLCT. The data in 19 relative to 1pRLC19.By applying the above methods, we next evaluated the changes in phosphorylation of RLC that occur upon stimulation with OT and Calp treatments in the presence of cell-permeable inhibitors of ROK and MLCK  to determine if phosphorylation at S1 was inducible by PMA. 2+-phos-tag method. Subsequent attempts to improve resolution of the unidentified bands from 0pRLC, 1pRLC and 2pRLC by Mn2+-phos-tag were inconsistent. Recently, an improved method of separation using Zn2+ instead of Mn2+ ions in the phos-tag matrix and an alternate buffer set for electrophoresis was developed 2+-phos-tag method to study the PMA-induced changes in the RLC phospho-states. In order to detect as many phospho-states as possible, we studied the effects of PMA and of the protein phosphatase 1 and 2A inhibitor, calyculin A. To elucidate the phosphoisotypes contained within each phospho-state, we used the MMAb directed toward p19RLC since it is detected independently using a secondary Ab directed towards murine primary Ab whereas the CtRLC and p1RLC (both RPAbs) are detected using a secondary Ab directed towards rabbit polyclonal Abs. Thus far, we have focused on the alkaline MnIn the present work, we treated three phospho-states of RLC as a closed system based on strong evidence that other states are unlikely to exist physiologically. This rationale is in agreement with our data ,3–5 andIn our early experiments, we assumed that all phosphoisotypes with equivalent phosphate stoichiometries comigrate in the phos-tag acrylamide matrix. However, the data in in vitro data suggest that PKC-mediated phosphorylation of RLC results in reduced myosin ATPase activity and impaired S19 phosphorylation by MLCK in vitro motility assay for myosins isolated from various sources demonstrated that PKC phosphorylation of RLC could not induce movement of myosin beads along actin cables on its own, and also did not alter the rate of movement of the myosin beads after MLCK-mediated phosphorylation of RLC Some The protocol to obtain biopsies from the lower segment of the uterus at the time of cesarean section was reviewed and approved by the Human Ethics Review Board of the University of Alberta and the Ethics Review Board of Alberta Health Services, the provider of healthcare services in the region. A research nurse at the Royal Alexandra Hospital in Edmonton obtained informed and written consent from each patient.g for 5 min, and washed twice with PBS. Isolated uterine myocytes were grown in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% antimycotic/antibiotic (Gibco) at 37°C in humidified 5% CO2/95% air. Cell cultures were grown to 80–100% confluence in a single well of a 6-well plate post-isolation, then in T25 and T75 flasks . Prior to experiments, the myocytes are seeded at 600 cells/mm2 in 6-well plates and allowed to adhere and equilibrate O/N. The next morning, the cells were washed once with pre-warmed DMEM containing no additives, and then placed into the incubator in DMEM without additives for 2–4 hrs before treatment. All stimulants, pharmacologic agents, and drug diluents  were diluted in DMEM without additives to the final concentration prior to experiments. Oxytocin , glycyl-H-1152 , ML7 , and phorbol-12-myristate-13-acetate  were obtained from Calbiochem . 4-α-PMA  was purchased from Santa Cruz Biotechnology Inc. . Calpeptin  and the cell permeable rhoA inhibitor  were purchased from Cytoskeleton, Inc . Calyculin A  was purchased from Cell Signaling . Uterine myocytes were treated for 20 sec with OT, 30 min with g-H, ML7, PMA, 4-α-PMA, or Calyculin A and 15 min with Calp, as determined by optimization experiments.This methodology has been detailed previously 2O), recovered into microtubes, and centrifuged at 18,000 g for 20 min. The protein pellets were washed several times with acetone-DTT (10 mM), allowed to air dry, then resuspended in 75 µL of 1× SDS-loading buffer by boiling for 10 min. 15 µL of each sample was loaded per lane (approximately 25 µg protein/lane) on Tris-Glycine-SDS minigels. Electrophoresis was carried out by standard methods, in 15% acrylamide gels. In addition, two methods of phos-tag  according to the manufacturers’ specifications. Briefly, uterine myocytes were treated with 0.5 mU/mL Calp for 15 min, and then harvested by lysis in ice-cold buffer (provided). An aliquot of these samples was retained for protein content estimation by ADV02 reagent , and the rest was flash frozen in N2(l), and stored at −80°C until needed. The frozen samples were thawed and prepared for the assay as specified. Approximately 25–30 µg total protein was used per well, and each sample was assayed in duplicate. The measured optical density measured at 490 nm was expressed relative to vehicle control. These data were normalized for total-rhoA content by performing WB analysis in parallel with the rhoA activity assay. Preliminary experiments comparing uterine myocyte lysates to purified, constitutively active rhoA (G14V) positive controls demonstrated that our measurements were within the linear range of the assay.Relative rhoA activity was estimated by a commercially available ELISA-based assay (‘G-LISAAll results were expressed using means ± S.E.M. In-cell western data were analyzed using one-way ANOVA, followed by a post-hoc Tukey test. Two-tailed independent t-tests were used to analyze rhoA activation and WB data, and phospho-RLC proportion data in


The ribosome’s interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5′ untranslated regions (5′ UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome’s interactions with structured standby sites, upstream of Shine–Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA’s translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome’s binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome’s translation initiation rates for 136 synthetic 5′ UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical mechanism for observed context effects and long-range post-transcriptional regulation. Changesr ligand . Howeverr ligand  and are r ligand , many nar ligand A. Here, Supplementary Figure S1), followed by coordinated insertion of the mRNA into the Entry channel and the 16S rRNA’s base pairing with SD like sequences to position the start codon over the P-site   ,24–28.in vivo reporter assays. We use thermodynamic modeling to quantify how the ribosome binds less accessible 5′ UTRs, accommodates or partially unfolds RNA structure, selects from multiple binding sites, slides across the mRNA and initiates translation. We develop a predictive biophysical model that combines thermodynamic minimization with a four-parameter free energy model to accurately predict the translation initiation rates of 136 long, diversely structured 5′ UTRs. Finally, we demonstrate that the ribosome can bind to distant sites and engage in long-range RNA interactions, providing a mechanism for the observed context effects and new ways for riboswitches and small RNAs to regulate translation.In this article, we decipher the biophysical rules governing the ribosome’s interactions with long, structured 5′ UTRs. We systematically alter 5′ UTR sequence, structure and shape to measure the ribosome’s interactions using R) (70 constitutive promoter (BioBrick #J23100), a synthetic 5′ UTR and a codon-optimized mRFP1 fluorescent protein. Synthetic 5′ UTRs were inserted by standard cloning techniques, utilizing either BamHI and SacI, or XbaI and SacI sites, depending on 5′ UTR length. 5′ UTRs with desired sequences were created by either annealing of complementary oligonucleotides with overhangs, or by PCR assembly of oligonucleotides with corresponding restriction sites. Plasmid variants were cloned by restriction digest of DNA backbone, purification and ligation to DNA fragments with 5′ UTR sequences and transformation of chemically competent Escherichia coli DH10B cells via heat shock. Correct clones were identified by DNA sequencing. All the designed 5′ UTR sequences are presented in the Supplementary Data.Luria-Bertani (LB) media  was obtained from BD and supplemented with 50 µg/ml chloramphenicol (Sigma-Aldrich). The expression system is derived from the pFTV1 plasmid   and encoE. coli DH10B cultures in an alternating pattern that excluded the outer wells. Cultures were incubated overnight at 37°C with 200 rpm orbital shaking. A fresh 96-well plate containing 200 μl LB and chloramphenicol media was inoculated by overnight cultures using a 1:100 dilution. Plates were incubated at 37°C in a plate-based spectrophotometer (TECAN M1000) with high orbital shaking. OD600 measurements were recorded every 10 min. Once a culture reached an OD600 of ∼0.15, a sample of each culture was transferred to a new plate containing 200 μl phosphate buffered saline  and 2 mg/ml kanamycin (Sigma Aldrich) for flow cytometry measurements. Periodic serial dilutions were repeated two more times, maintaining cultures within the exponential growth phase. Three samples were taken from each culture. The single-cell fluorescence distributions of samples were measured using a Fortessa flow cytometer (BD Biosciences). All distributions were unimodal. The autofluorescence distribution of E. coli DH10B cells was also measured. The arithmetic mean of each distribution was taken, and the mean autofluorescence was subtracted from each sample. The reported fluorescence values in this study are the average of three measurements (n = 3).Fluorescent protein reporter measurements were performed in 96-well format. An initial 96 deep-well plate containing 700 μl LB and 50 μg/ml chloramphenicol was inoculated, from single colonies, with up to 30 different Ct calculations were then averaged for each sample.For mRNA level measurements, single colonies were first grown overnight in 5 ml LB media with chloramphenicol, then diluted to 0.01 OD600 in fresh media and grown to mid-exponential phase until reaching 1.0 OD600, as measured by a cuvette-based spectrophotometer (NanoDrop 2000C). Total RNA was extracted using the Total RNA Purification kit (Norgen Biotek). Treatment with Turbo DNAse (Ambion) eliminated genomic DNA contamination. Reverse transcription was carried out with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems), followed by quantitative PCR with a TaqMan probe (5′-ACCTTCCATACGAACTTT-3′), targeting the internal coding sequence for mRFP1 (forward primer 5′-ACGTTATCAAAGAGTTCATGCGTTTC-3′ and reverse primer 5′-CGATTTCGAACTCGTGACCGTTAA-3′) and using a ABI 7300 real-time cycler. A TaqMan probe for 16S rRNA was used as an endogenous control. Triplicate measurements were each performed on two separate RNA extractions. ΔΔThe complete biophysical model employs a free energy model to calculate the total binding free energy between the ribosome and mRNA, according to the equation C:(1)r, according to:Using statistical thermodynamics, we may relate this total Gibbs free energy change to the mRNA’s translation initiation rate, Gtotal varied from −10 to 16 kcal/mol, resulting in well-predicted translation rates that varied by over 100 000-fold . After assembly of the 30S ribosomal subunit, the last nine nucleotides of its 16S rRNA have hybridized to the mRNA, while all non-clashing mRNA structures are allowed to fold. The free energy of folding for this mRNA–rRNA complex is ΔGmRNA–rRNA (ΔGmRNA–rRNA is negative). mRNA structures that impede 16S rRNA hybridization or overlap with the ribosome footprint remain unfolded in the final state. These Gibbs free energies are calculated using a semi-empirical free energy model of RNA and RNA–RNA interactions . The 30S ribosomal subunit also prefers a five nucleotide aligned spacing (Gspacing > 0) and aligned spacing distance was measured in our previous study . Hairpins are only considered as components of standby site modules if they are mutually exclusive with the 16S rRNA binding site. A 5′ UTR with multiple upstream hairpins will contain multiple standby site modules. We consider a 5′ UTR without upstream hairpins to contain a single standby site module with zero hairpin height and zero distal site length. Using these definitions, the biophysical model calculates a ΔGstandby for each standby site module.The biophysical model automatically decomposes a 5′ UTR into one or more standby site modules using the following definitions. A standby site module contains a single mRNA hairpin, surrounded by two single-stranded regions: an upstream distal binding site and a downstream proximal binding site. The lengths of the distal and proximal binding sites are denoted by As, of a standby site module is determined according to As = 15 + P + D – H. The key aspect of this equation is that proximal length, distal length and hairpin height have equal and interchangeable effects on the amount of single-stranded surface area. The constant 15 was selected to create a positive metric for single-stranded surface area. Based on data shown in H in the equation is not allowed to exceed 15 nt.The available single-stranded surface area, As,  reject the null hypothesis, showing that the differences in two sets of model predictions were statistically significant.Two-sample H), upstream distal binding site length (D) and downstream proximal binding site length (P) . Changes in fluorescence are primarily due to changes in translation rate and, in these growth conditions, fluorescence and translation initiation rates are proportional. All sequences and measurement data are listed in the Supplementary Data.We first employed a learn-by-design approach to determine the ribosomal platform’s interactions with standby sites that contain a single hairpin, and therefore contain a single standby site module. Standby site modules are defined by their hairpin height (ngth (P) . All mRNngth (P) C–E. In cSupplementary Data, column E). Highly accessible standby site modules with long proximal binding sites or short hairpins also cause the mRNA’s translation rate to reach a plateau with the same fluorescence level as an mRNA with an unstructured 5′ UTR  are interchangeable, and that its available single-stranded surface area, As, determines the ribosomal platform’s ability to bind to it.According to the data, standby site module accessibility plays an important role in modulating the ribosome’s interactions with 5′ UTRs and controlling translation rate across a large range. In particular, lengthening the distal binding site by four nucleotides increased the mRNA’s translation rate by 4.2-fold, whereas lengthening the proximal binding site by the same amount increased it by 4.0-fold. Shortening the hairpin’s height by 4 bp increased the mRNA’s translation rate similarly by 3.1-fold. More generally, the slopes (first derivatives) of the log-transformed translation rates all have similar magnitudes  (standby = 0), indicating that the mRNA’s translation initiation rate is not reduced by the ribosomal platform’s interactions. A standby site module with a positive binding free energy penalty (ΔGstandby > 0) has reduced the mRNA’s translation initiation rate by decreasing the ribosomal platform’s ability to initially bind the mRNA.We now turn to thermodynamic modeling to quantify the ribosome’s interactions with standby site modules. The competitive binding of free ribosomes to the pool of intracellular mRNAs allows one to use statistical thermodynamics to relate a ribosome’s binding free energy to its translation initiation rate, according to a constant log-linear relationship  13,14).,14.13,14cal/mol) F. A maxiSupplementary Data). For branched structures, we show that the relevant hairpin height is the average across the multiple branches  . The quadratic relationship accurately predicted the ribosome’s translation rate and binding free energy penalty , confirming that standby site module’s accessibility controls the ribosome’s ability to bind and initiate translation . Unfolding a single base pair in a module’s hairpin will increase the standby site module’s surface area by three nucleotides, the hairpin’s height decreases by one and the lengths of both the proximal and distal binding sites increase by one. Therefore, unfolding a module’s hairpin will lead to higher accessibility, a lower ΔGdistortion and a higher translation rate. However, unfolding base pairs requires an input of free energy (ΔGunfolding > 0), which will lead to a lower translation rate. Consequently, there is an energetic trade-off between unfolding mRNA structures, and increasing the surface accessibility of standby site modules. The ribosome may completely unfold a structured standby site module to maximize its surface area, but the energetic cost may be high . In contrast, the ribosome may fully accommodate a standby site module’s hairpin, without unfolding it, even though its low surface accessibility will penalize the ribosome’s ability to bind to it .By definition, there is a coupled thermodynamic relationship between the unfolding of an mRNA hairpin and increasing the accessibility of the corresponding standby site module, where the accessibility of standby site module controls the ribosomal platform’s distortion energy penalty ΔGstandby = ΔGdistortion + ΔGunfolding). P = 2 nt), a short distal binding site (D = 2 nt) and a long hairpin (H = 15 nt), resulting in a largely inaccessible 5′ UTR (As = 4). Based on the standby site module’s surface area, the ribosome has an initial distortion energy penalty of 11.5 kcal/mol. However, the ribosome can selectively unfold the hairpin’s closing A:U base pairs, increasing the standby site module’s surface area and lowering its ΔGdistortion at the expense of increasing its ΔGunfolding. Unfolding 1 bp increases the standby site module’s surface area by 3 nt, decreases the distortion energy penalty to 7.8 kcal/mol and increases the unfolding energy penalty to 0.90 kcal/mol, leading to a total binding free energy penalty of 8.7 kcal/mol. The ribosome can continue to unfold base pairs to minimize its total binding free energy penalty; unfolding 3 bp at a cost of 2.7 kcal/mol leads to a distortion energy penalty of 2.6 kcal/mol, and a total binding free energy penalty of 5.3 kcal/mol . Importantly, thermodynamic minimization does not add any new parameters to the model.Based on thermodynamic principles, we hypothesize that the ribosome will ‘selectively’ and ‘partially’ unfold structured standby site modules to minimize its total binding free energy penalty  and differing hairpin sequences, sizes and folding energetics . We employed the minimization principle to calculate the number of unfolded base pairs and to predict the ribosome’s binding free energy penalty, ΔGstandby (Gdistortion and ΔGunfolding = 0) (Gunfolding and ΔGdistortion = 0) (We tested the proposed mechanism by characterizing the translation rate of 22 synthetic 5′ UTRs C–E that Gstandby D. As coming = 0) C or thation = 0) E.Gstandby), rather than separately minimize its distortion or unfolding penalties . Remarkably, in all 22 cases, only 1–3 bp of each hairpin are predicted by the model to be unfolded, while accommodating the remaining large RNA structures.The data supports our hypothesis that the ribosome partially and selectively unfolds structured standby site modules to minimize its total binding free energy penalty  with up to four standby site modules to investigate the potential for cooperative binding A. If theWe found that 5′ UTRs with two, three or four standby site modules were all translated at the same rate, indicating an absence of cooperative binding B, includGstandby between 9.0 and 17.4 kcal/mol) compared to the maximally accessible upstream standby site modules. Therefore, if the ribosome binds the farthest upstream standby site module, its translation rate will be at least 12.7-fold higher than binding to the internal standby site modules. We measured fluorescence levels with flow cytometry and mRNA levels with RT-qPCR. Compared to an unstructured 5′ UTR, the mRNA levels decreased by only 1.3- to 5.5-fold, while the fluorescence levels were correspondingly reduced between 2.5- and 18.3-fold, showing that changes in fluorescence were mainly due to changes in translation rate and ribosome binding free energy penalties  E. In parSeveral mechanisms would allow the ribosomal platform to bind distant, upstream standby site modules and transition to a pre-initiation complex at the start codon. After the ribosome has bound to the upstream standby site module, the 30S complex could engage in diffusive hopping that would increase its local concentration and assembly rate at the start codon. The rate of assembly would largely depend on the physical distance between the standby site module and start codon, and not on the presence of RNA structures. Alternatively, the ribosome could employ a full contact, sliding mechanism that reorients the mRNA until a stable pre-initiation complex has formed. The positively charged surface of the ribosomal platform makes it ideal for maintaining contact with a 5′ UTR until 16S rRNA hybridization has anchored the mRNA. Unlike a diffusive hopping mechanism, the presence of mRNA structures would impede this sliding motion and reduce the pre-initiation complex’s assembly rate.Further analysis of our measurements shows that RNA structures in between the upstream standby site module and start codon partly attenuate the mRNA’s translation rate E. The apc = 0.2 kcal/mol/nt) to yield a free energy penalty of sliding, ΔGsliding. The sliding free energy penalties were 0, 3.0, 4.8 and 7.8 kcal/mol for M1 to M4 5′ UTRs, respectively. We add together the sliding, distortion and unfolding free energy penalties to calculate the ribosome’s binding free energy penalty to each standby site module.Our data supports the presence of a sliding mechanism for the ribosomal platform. Intervening mRNA structures are not unfolded, but are pushed aside with an energetic cost. Using our measurements of the M2 5′ UTR, we estimate that 3.2 ± 0.5 kcal/mol free energy is required to push aside the single RNA structure with a height of 15 nt, yielding a coefficient of 0.20 ± 0.04 kcal/mol per hairpin height. We then determined whether including this sliding penalty could improve the model’s ability to predict the translation rate of long 5′ UTRs with multiple standby site modules with ‘hairpins of differing heights’. For each standby site module, we calculate the heights of all downstream RNA structures, and multiplied the sum by the coefficient .Altogether, the ribosome binds to 5′ UTRs by selecting the most geometrically accessible standby site module that requires the least amount of RNA unfolding. Standby site module selection is not limited by distance to the start codon, and upstream standby site modules can support high rates of translation. We identified that ribosomal distortion, selective RNA unfolding and ribosomal sliding control standby site module selection and binding energetics, and combine them together to create a single biophysical model. The ribosome’s binding free energy penalty to the standby site modules in a structured 5′ UTR is calculated according toGstandby = 0 kcal/mol). All RNA energetics are calculated using a semi-empirical free energy model of RNA and RNA–RNA interactions . The biophysical model was able to accurately predict the relative translation rates and ribosome binding free energy penalties  (R2 = 0.89)  with two to four standby site modules of varying geometries, RNA structure energetics and hairpin heights ( = 0.83) A. Overal = 0.89) B.FigureE. coli MG1655 genome, provided by EcoCyc , making them targets for translation activation. Moreover, 79 diverse 5′ UTRs with an average length of 175 nt have positive sliding energy penalties (ΔGsliding), indicating that distant standby site modules are controlling translation, and making them potential targets for long-range regulation.The model predicts that both types of translation regulation occur in natural 5′ UTRs. In particular, 39% of genomic 5′ UTRs have standby site modules whose distal or proximal binding sites are longer than 15 nt, which are potent targets for translational repression; in contrast, 9% of 5′ UTRs contain standby site modules with low accessibilities . To offset this large energetic burden, model calculations indicate that the ribosomal platform partially unfolded 6 bp from the domain 2 hairpin, which minimized its binding free energy penalty to a ΔGstandby of 5.32 kcal/mol . Destabilization of the domain 2 hairpin (ILOΔ5) restored the mRNA’s translation to its wild-type rate by increasing the surface area of the standby site to 20.5 nt, resulting in an almost zero ΔGstandby. The biophysical model calculations indicate that other mutations to the thrS 5′ UTR  to its available single-stranded surface area, implicitly using coefficients of one to indicate interchangeability. Additional measurements will be needed to precisely measure these coefficients, and relate them to the helical shape of A-form RNA. In addition, tertiary structures, such as G-quadruplexes and pseudoknots, will likely have a differently defined metric of surface area. Further investigation will be necessary to determine the effect of tertiary RNA structures on standby site accessibility, ribosome binding and translation initiation.In this study, we introduce a new metric to quantify the surface area of a standby site module, which was validated using diverse, but canonical, RNA secondary structures. The metric relates the geometric characteristics of the standby site module . Thirteen outliers have errors between 2 and 4 kcal/mol (Supplementary Data). Some outliers are due to the effects of increased mRNA degradation of 5′ UTRs with very long proximal or distal binding sites  . In others, extremely short proximal or distal binding sites can result in non-canonical base pairings that alter the ribosome’s binding affinity. For example, in the absence of a proximal binding site (P = 0 nt), co-axial stacking will take place between the nucleotides in the distal binding site and the mRNA–rRNA duplex that anchors the ribosome to the mRNA. Similarly, co-axial stacking will play a larger role in predicting the effects of small RNAs that bind to sites nearby RNA structures.Analysis of the model’s error can lead to further insight into the ribosome’s interactions. The model’s predictions have a normally distributed error for 90% of sequences (http://salis.psu.edu/software.As the list of interactions that control gene expression continues to grow, we will stretch the limits of human pattern recognition and the use of observational correlations. Biophysical modeling offers a comprehensive approach to account for all known interactions with the gene expression machinery, identify gaps, design experiments, precisely measure strengths and make testable predictions. The accuracy of these models will go hand-in-hand with our ability to engineer genetic systems without trial-and-error. We have incorporated the ribosome’s interactions with structured standby site modules into an improved biophysical model, called the RBS Calculator v2.0. A software implementation and user-friendly web interface is available at Supplementary Data are available at NAR Online.


Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Here, we identified a Haspin kinase, designated AtHaspin, in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in  The mitotic phase, which comprises mitosis and cytokinesis, is a fundamental process for faithful transmission of genetic information from one cell generation to the next. The main purpose of mitosis is to segregate sister chromatids into two daughter cells. The regulation of mitotic progression relies mainly on two post-translational mechanisms; protein phosphorylation and proteolysis. Cell division is regulated by mitotic kinases, such as the cyclin-dependent kinase 1 (CDK1), the Polo family, the NIMA (never in mitosis A), and the Aurora family, as well as kinases implicated in mitotic checkpoints, mitotic exit and cytokinesis [Arabidopsis thaliana Aurora kinase3) phosphorylates histone H3 at Ser10 and Ser28 in vitro [Arabidopsis suspension cells [Post-translational modifications of core histones play a crucial role in chromatin structure and gene expression . Althougin vitro . Inhibiton cells . Thus, AH3T3 and H3T11 are also phosphorylated, but their distribution patterns differ from those of H3S10ph and H3S28ph during mitosis. In mammalian cells, H3T3ph and H3T11ph occur preferentially at the centromere from prophase to anaphase ,14. In cSaccharomyces cerevisiae, Xenopus, and human revealed the novel cascade leading to the recruitment of mitotic kinases to the centromere [S. cerevisiae, Haspin interacts with cohesin, and the cohesin-associated Haspin phosphorylates histone H3 at Thr3 on the inner centromere [Haspin (haploid germ cell-specific nuclear protein kinase) was first identified as a testis-specific gene in mice ,18. Althntromere -24. In Sntromere . The phontromere -24. ThusA. thaliana Haspin, characterized its kinase activity, and determined its localization during mitosis. Expression of a kinase domain mutant of AtHaspin inhibited root growth, suggesting that Haspin is involved in cell division during mitosis.Analyses of Haspin were first carried out in yeast and animals, and although it is clear that this protein has roles in mitosis and cell division, the function of Haspin in organogenesis remains unclear. In this study, we identified Caenorhabditis elegans and S. cerevisiae, most organisms have one Haspin kinase gene. In a BLAST search of the A. thaliana genome, one Haspin candidate gene showed high similarity  to human Haspin kinase in the kinase domain. The second hit gene showed lower similarity . One Haspin candidate gene has been identified in some plant species, including ferns, mosses, and algae  is designated as AtHaspin . The C-terminal regions of Haspin proteins have a conserved kinase domain [2+ ion-binding sites were conserved between human and A. thaliana  (K309A) with or without ATP. Phosphorylated proteins were detected by ProQ Diamond Phosphoprotein Stain. As expected, GST-AtHaspin-KD was not autophosphorylated even in the presence of ATP , and plant histone H3 as the substrate. The positive control, GST-AtAUR3, is a histone H3 Ser10 and Ser28 kinase [in vitro. Surprisingly, GST-AtHaspin also phosphorylated histone H3 at Thr11 in vitro . Using anti-H3T3ph antibodies, bands were detected in the case of normal histone H3 and S10A, T11A, and G12A mutants, but not in the case of R2A, T3A, or K4A mutants  cultured cells with GFP-fused AtHaspin and observed tobacco BY-2 cells stably expressing AtHaspin-GFP with DNA stained by Hoechst 33342 . AtHaspin-GFP was expressed in meristems and primordia of root tips, lateral roots  using the estradiol-inducible XVE system . When gr Figures  and 6B.  Figures  and 7B.  Figures . These r Figures , and 7D. Figures . These rA. thaliana. Haspin kinases are conserved from yeast to human. Most organisms have one Haspin kinase gene, except for C. elegans and S. cerevisiae. Some plant species, including A. thaliana, have one Haspin candidate gene  and Ser28 (A25R26K27S28) [1R2T3K4) differs from that around Thr11 (K9S10T11G12). Point mutational analysis of human Haspin revealed the important residues for histone H3 phosphorylation in the catalytic cleft [A. thaliana . The human Haspin mutant D709N shows reduced affinity for the histone H3 tail and impaired ability to phosphorylate H3 [in vitro [in vivo. However, the centromeric localization of Dlk/ZIP during mitosis suggests that this kinase is responsible for H3T11ph. The A. thaliana genome contains no Dlk/ZIP kinase orthologues, and thus, AtHaspin has an additional role as a H3 Thr11 kinase in A. thaliana.As expected, GST-AtHaspin phosphorylated histone H3 at Thr3 in vitro ,22,24. I6K27S28) . Howeveric cleft . These ra Figure , green, ylate H3 . Althougin vitro , but theMitotic phosphorylation of histone H3 at Ser10, Ser28, Thr3, and Thr11 is highly conserved among eukaryotes. Although phosphorylation of histone H3 was first observed more than 30 years ago, the functions of this modification remain unclear . There iA. thaliana  was localized in the cytoplasm during interphase until NEBD. The interphase localization of AtHaspin differs from that of human Haspin, which localizes in the nucleus during interphase . After Nin vitro kinase assay revealed that the antibodies against H3T3ph and H3T11ph did not show cross-reactivity with other histone H3 phosphorylations  [Arabidopsis and BY-2 cells  . MAPs has Figure  and 4. Ts Figure . Furthers Figure . AlthougA. thaliana was identified as a mitotic histone H3 threonine kinase. The expression and dominant-negative analysis showed that AtHaspin may have a role in cell division during mitosis. The functions of H3 threonine phosphorylation remain obscure in animals and plants. Recent studies have shown that H3T3ph by S. cerevisiae, Xenopus and human Haspin is required for the accumulation of Aurora B on the centromere, and for subsequent activation of Aurora B kinase activity [A. thaliana [In this study, the Haspin kinase in activity -24. Theractivity ,41, homoactivity -24. The thaliana ,10-12,42A. thaliana (ecotype Columbia) seeds were grown on plates containing half-strength Murashige and Skoog salts, Gamborg B5 vitamins, 0.05% MES-KOH (pH 5.8), and 1% agar in a growth chamber at 22°C under continuous light. Tobacco 'Bright Yellow-2' (BY-2) cells were maintained as previously described by Kurihara et al. [a et al. .AtHaspin was cloned into the pDEST15 expression vector by Gateway technology (Invitrogen). Site-directed mutations of GST-AtHaspin for K309A and of the GST-H3 tail for R2A, T3A, K4A, S10A, T11A, and G12A were generated by PCR-based mutagenesis from the GST-AtHaspin and GST-H3 tail expression vectors, as described elsewhere [A full-length cDNA for the candidate Haspin kinase (RAFL15-11-J01) was obtained from the RIKEN BioResource Center . Site-dilsewhere .AtHaspin cDNA was cloned into the spUC-GFP vector, which contains the CaMV 35S promoter. spUC-AtHaspin-GFP was digested and ligated with pEBis-kH2 as described by Fujimoto et al. [o et al. .AtHaspin cDNA was cloned into the spUC-tdTomato vector [For inducible expression constructs, o vector . For Veno vector . PCR frao vector .AtHaspin native promoter, the promoter and genomic regions were amplified from A. thaliana genomic DNA. PCR products were cloned into pENTR using a pENTR/D-TOPO cloning kit (Invitrogen), and then into the pGWB4 expression vector by the LR reaction of the Gateway Cloning System (Invitrogen).For construction of the GFP expression vector driven by the in vitro kinase assay was performed with purified GST-AtHaspin or GST-AtHaspin-KD, the GST-histone H3 tail, and mutants as substrates as previously described by Kurihara et al. [The a et al. .Agrobacterium-mediated methods as described previously [Transformation of BY-2 cells and BY-GT16 cells  was carreviously . Stable eviously .Agrobacterium-mediated Arabidopsis transformation [Arabidopsis plants expressing AtHaspin-GFP driven by the native promoter were observed using an upright fluorescence microscope , or FluoView FV1000 (Olympus). Cell walls were stained with 10 μg/ml propidium iodide (PI).The floral-dip method was used for ormation . TransgeFor induction of AtHaspin-Venus and AtHaspin-KD-Venus, 10 μM 17-β-estradiol (Sigma) was added to MS medium  to induce expression during seed germination in sterile conditions. The root length of vertically grown seedlings was measured with MBF ImageJ software.The size of root meristems was determined by the position of the first endoduplicated cells, which were identified on the basis of their DNA content. Roots were fixed with 4% paraformaldehyde and 0.1% Triton X-100 for 40 min and stained with CyStain staining solution containing DAPI (Partec) for 5 min. Images were acquired using the IX-81 fluorescence microscope with a 0.5-μm section at the Z axis. These 2D images were used to construct 3D deconvolution images using AutoQuant X (Media Cybernetics). Cell ploidy was calculated using Metamorph (Molecular Devices). When three cells with more than 4C DNA content were aligned continuously, the top cell was defined as the first endoduplicated cell. Cell wall patterning was analyzed using the mPS-PI method . PI-staiANOVA: analysis of variance; BLAST: basic local alignment search tool; BY-2: Bright Yellow-2; CPC: chromosomal passenger complex; DAPI: 4',6-diamidino-2-phenylindole; EST: expressed sequence tag; GFP: green fluorescent protein; GST: glutathione S-transferase; H3S10ph: phosphorylation of histone H3 at Ser10; H3S28ph: phosphorylation of histone H3 at Ser28; H3T3ph: phosphorylation of histone H3 at Thr3; H3T11ph: phosphorylation of histone H3 at Thr11; KD: kinase dead; MAP: microtubule-associated protein; NEBD: nuclear envelope breakdown; PFA: paraformaldehyde; PPB: pre-prophase band; QC: quiescent center.DK conceived and designed the study, carried out the experiments, performed the statistical analyses, and wrote the manuscript. SM participated in the design of the study, carried out the ploidy analysis and the observations of AtHaspin-KD overexpression in root tips, and revised the draft of the manuscript. TO carried out the phenotypic analyses of plants overexpressing AtHaspin-KD. TH and KF participated in the coordination of the study. All authors read and approved the final manuscript.Dynamics of AtHaspin-tdTomato and GFP-α-tubulin in tobacco BY-2 cells. Dynamics of AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green) from late G2 phase to early G1 phase in BY-2 cells after 48-h induction with 10 μM 17-β-estradiol. Images were acquired at 30-s intervals under a 40 × objective lens, and movie is displayed at 15 frames per second (fps). Numbers indicate time (h: min).Click here for fileArabidopsis root tipsDynamics of AtHaspin-GFP in . Images were acquired at 1-min intervals under a 40 × objective lens, and movie is displayed at 15 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileArabidopsis torpedo embryoDynamics of AtHaspin-GFP in . Images were acquired at 1-min intervals under a 40 × objective lens, and movie is displayed at 10 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileArabidopsis root tipsDynamics of AtHaspin-GFP in . Images were acquired at 90-s intervals under a 100 × objective lens, and movie is displayed at 5 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileAbnormality of cell orientation in AtHaspin-KD plants. At 6 days after imbibition, Col. plants and transformants with inducible AtHaspin-KD-Venus vectors with or without induction were analyzed using the mPS-PI method. In AtHaspin-KD plants, arrows indicate abnormalities in orientations of cell walls between transverse neighboring cells. Scale bar: 50 μm.Click here for file


B. Stress perception increases the phosphorylation of the stressosome sensor protein, RsbR, and the scaffold protein, RsbS, by the protein kinase, RsbT. Subsequent dissociation of RsbT from the stressosome activates the σB cascade. However, the sequence of physical events that occur in the stressosome during signal transduction is insufficiently understood.The stressosome is a bacterial signalling complex that responds to environmental changes by initiating a protein partner switching cascade, which leads to the release of the alternative sigma factor, σHere, we use computational modelling to correlate the structure of the stressosome with the efficiency of the phosphorylation reactions that occur upon activation by stress. In our model, the phosphorylation of any stressosome protein is dependent upon its nearest neighbours and their phosphorylation status. We compare different hypotheses about stressosome activation and find that only the model representing the allosteric activation of the kinase RsbT, by phosphorylated RsbR, qualitatively reproduces the experimental data.Our simulations and the associated analysis of published data support the following hypotheses: (i) a simple Boolean model is capable of reproducing stressosome dynamics, (ii) different stressors induce identical stressosome activation patterns, and we also confirm that (i) phosphorylated RsbR activates RsbT, and (ii) the main purpose of RsbX is to dephosphorylate RsbS-P. Bacillus subtilis is activated in response to diverse environmental stresses, including ethanol, temperature, UV light, and osmolarity, and initiates a protein partner switching cascade that leads to the release of the alternative transcription factor σB (. (2004) ). The au  [et al. (2004) [In the Kim et al. 04 20] s set al. B operon gene expression levels. Therefore, while we captured the ranges of RsbS and RsbR phosphorylation, the dynamics of the RsbS deactivation process are not reproducible in our framework. A notable difference between the models was the faster activation of RsbR and RsbS in the ‘no-cooperation’ model in Figure et al. (2004) [We did not attempt to model the long-term regulation of stressosome activation because that requires the additional consideration of changes in the σe et al. 04 [20], et al. (2008) . Only if the phosphorylation probability was smaller than the random number was phosphorylation deemed to have occurred. Dephosphorylation was determined similarly using the dephosphorylation parameter. Simulations were repeated 50 times while assuring that statistical properties did not change significantly. The model was implemented in Matlab(R) (7.11.0) and is available as Additional file In the simulation the initial phosphorylation state of RsbR and RsbS was randomly assigned with a probability of 50% for each to allow rapid equilibration of the system. The equilibrium was independent of the exact initial state which affects the relaxation time only. During a simulation step we determined for all 60 proteins, in random order, whether a phosphorylation reaction would occur or not. For instance, the triangle R2 has neighbours  with a phosphorylation status {0,1,1} and the central RsbR is non-phosphorylated. From Table et al. (2008) [The experimental data by Marles-Wright . (2008)  and our a represents the maximum response, the β-galactosidase activity in the experiments  and RsbS phosphorylation in the simulation (a=0.2 for ‘product activation’). Parameter b encodes the sigmoidality, i.e. how fast the system switches between on and off (b=8*10-1/6*10-3 for ethanol/NaCl and b=12 for ‘product activation’). Parameter c encodes the inflection point; in the experiments this is the concentration of stressor producing half maximal β-galactosidase activity , in the simulation this is the RsbR phosphorylation probability resulting in half maximal RsbS level (c=0.14 for ‘product activation’). The response  were divided by their associated estimated a-parameter in the hyperbolic tangent formula. For the experiment both signals, i.e. NaCl and ethanol concentrations, were divided by their respective c parameter. For the simulation the signal parameter, kphr , was divided by its associated c parameter. Thus, all data in the response range from zero to approximately one, and the response of 0.5, correlates to signal strength 1.In Equation 1, B. subtilis: Bacillus subtilis; UV light: Ultra-violet light; cryo-EM: Cryo-electromicroscopy; Rsb: Regulator of σB; ADP: Adenosinediphosphate; ATP: Adenosinetriphosphate.The authors declare no conflict of interest.UWL initiated the concepts for the study, performed modelling and simulation, and analysed the data. TM participated in the modelling and simulation, JMW, RJL and OW contributed to the conception and analysis. All authors edited, read, and approved the final manuscript.Figure S1. Akbar et al. (2001) [B. subtilis  [kphs. The open circles represent the wildtype with all kphs are RsbD stressosomes  [kphs to 0.75 . Akbar et al. (2001) [. (2001)   . The sim., 2010) , is to i. (2001)  left), r. (2001)  measuredClick here for fileThe Additional file 2 contains the matlab files for reproduction of the results. To use it unpack all files into one directory. The main file is called liebal_stressosome-model_12_workflow-matlab.m. it is written using cell-mode. The cells can be evaluated sequentially by setting the cursor in a cell and evaluating it, eg. by Ctrl+Enter. Please contact ulfliebal@gmail.com for any questions.Click here for file


Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1. Receptor-mediated endocytosis is a process by which cells internalize extracellular ligands. It is initiated by the inward budding of plasma membrane vesicles containing ligands bound to specific transmembrane receptors. Among other routes, receptors can be internalized via clathrin-coated pits that pinch off, lose their clathrin coat and fuse with an endosomal compartment where the cargo is sorted towards recycling or degradation in lysosomes. Several endosomal populations can be distinguished based on the presence of specific markers as well as functional and morphological characteristics. Early endosomes that contain the small GTPase Rab5 and EEA1 (early endosome antigen 1) are the first cargo sorting platform Ubiquitination acts as a signal for the internalization and sorting of plasma membrane proteins along the endocytic route , contributing to signal transduction downstream of these receptors , and are preferentially localized underneath the plasma membrane While proteasomal inhibition affects endocytic trafficking already upon short application (1 h or less), prolonged (12–24 h) treatment causes massive changes in the turnover of cellular proteins. Proteasome deficiency stimulates a cellular stress response by inducing time- and dose-dependent inhibition of cell growth, arrest of the cell cycle, apoptosis, as well as the loss of mitochondrial membrane potential and increase in the intracellular ROS levels Here, we aimed to characterize an effect of proteasomal inhibition on APPL endosomes. We analyzed the localization and morphometric features of APPL1-positive endosomes upon a range of incubation times with proteasome inhibitors. Strikingly, we observed initial solubilization of APPL1 from endosomal membranes followed by its relocalization to aggresomes which correlated with increased ubiquitination and insolubility of APPL1.Anti-APPL1 polyclonal antibodies against C-terminal peptides were raised in rabbits (Eurogentech) and previously described The constructs of untagged, Myc-tagged and GFP-tagged APPL1 were previously described We used two double stranded siRNA oligonucleotides against human APPL1 from HP GenomeWide siRNA collection (Qiagen): (APPL1-a) Cat. No. SI02652125, (APPL1-b) Cat. No. SI03128979. Alongside, two siRNA negative controls were used (Qiagen): (control-a) Cat. No. 1027280 and (control-b) Cat. No. 1022076.Human cell lines HEK293 (embryonic kidney) and HeLa (cervix carcinoma) were cultured as previously described HeLa cells were processed for immunofluorescence staining as previously described www.kalaimoscope.com) 2 (corresponding to the average vesicle size), and the total fluorescence of a fluorophore detected in all vesicles, defined as the total integral vesicle intensity. These parameters were calculated separately for red (APPL1) and green (EEA1) channels based on more than a hundred of imaged cells per condition.For morphometric image analysis we used the custom-designed image analysis software MotionTracking/Kalaimoscope  in order to view the entire cell volume.g for 15 min. The pellets were washed and boiled in SDS-PAGE sample buffer. Total cellular lysates were prepared by adding hot SDS-PAGE sample buffer to the dish with adherent cells, scraping and boiling prior to loading on the gel. Resolved proteins were transferred to nitrocellulose membrane (Whatman), probed with specific antibodies, and detected with enhanced chemiluminescence.Cells were lysed in RIPA buffer containing 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris–Cl (pH 7.4), 150 mM NaCl, 0.5 mM EDTA and protease inhibitor cocktail. Soluble lysate samples of 10–20 μg total protein were subjected to SDS-PAGE. Protein concentration was measured with Bradford assay (Roth). Where appropriate, insoluble fractions were prepared from cellular pellets formed upon extraction for 20 min on ice with RIPA buffer, followed by centrifugation at 20,000Proteins of interest were immunoprecipitated from soluble lysates containing 100–200 μg of protein by 2-h incubation with an appropriate antibody at 4 °C with constant rotation, recovery of immunocomplexes on protein A sepharose beads (Roche), washing in IP buffer , 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol), and elution with SDS-PAGE sample buffer. Where indicated, non-immune rabbit immunoglobulins were used in control immunoprecipitations.U test. The level of significance was set at p < 0.05. Analyses were performed using GraphPad Prism software.The statistical significance was assessed by using the Mann–Whitney In order to investigate the effect of proteasome inhibitors on APPL endosomes, we looked at the distribution of endogenous APPL1 in HeLa cells treated with MG132, ALLN, or bortezomib. Both MG132 and ALLN are cell-permeable peptide aldehydes commonly used as reversible inhibitors of the chymotrypsin-like proteolytic activity of the proteasome. Bortezomib (Velcade), a dipeptide boronic acid analogue, has increased affinity for the catalytic subunits of the proteasome and reduced multidrug resistance sensitivity. It represents a prototype of highly effective anti-cancer agents, used for treatment of two B-cell malignancies: multiple myeloma and mantle cell lymphoma In order to check whether the changes in APPL1 distribution upon prolonged proteasomal inhibition are specific, we looked at the localization of other proteins residing on early or late endosomes as well as at the distribution of other cellular organelles . We founIn order to quantify the extent of observed changes in APPL1 localization, morphometric features of APPL1- and EEA1-containing endosomes were compared using the image analysis software MotionTracking At the same time, the EEA1 endosomes displayed much less changes. The number of EEA1 endosomes was not affected by MG132 treatment A. The arBased on this data, we could conclude the following: (a) Inhibition of proteasomes decreased the number of detectable APPL endosomes by half, while it did not affect the number of observed EEA1 vesicles; (b) Mean size of APPL vesicles initially decreased and then returned to the control levels upon MG132 treatment, with a heterogeneous vesicle population after 20 h ; (c) MG132 treatment solubilized APPL1 from vesicles, as the massive decrease in the amount of APPL1 bound to vesicles could not be explained by the observed changes in the vesicle size; (d) MG132 treatment reduced the size of EEA1 vesicles and, proportionally, the amount of vesicle-bound EEA1. Clearly, MG132 affects both early endosomal compartments . However, the extent and timing of MG132 effects seem to be substantially different in the case of APPL1 and EEA1 endosomes. In the case of EEA1, MG132 treatment primarily affected the vesicle size. In the case of APPL1, changes were much more pronounced and involved the decreased amounts of vesicle-bound APPL1 molecules as well as the decreased number of detectable APPL1-positive vesicles.Although the morphometric image analysis provided us with quantitative data, due to averaging of analyzed parameters within endosomal populations it was not optimal for characterizing distinct patterns of APPL1 redistribution upon the two time periods of MG132 treatment. To complement the initial analysis, we counted the number of HeLa cells containing perinuclear clusters of APPL1 upon treatment with DMSO or MG132 for 4 or 20 h. Representative images are shown in At this point it was important to determine whether perinuclear clustering of APPL1 represents its recruitment to endosomes relocalized in the cell center or a formation of proteinaceous aggregates without a surrounding membrane. We checked this by employing continuous uptake of fluorescently labeled cargo during the incubation with MG132 for 18 h. We used transferrin that is constitutively recycled and EGF that undergoes degradation in lysosomes, both labeled with Alexa Fluor 488. In addition, the cells were stained for APPL1 and for vimentin in order to visualize aggresomes . These are proteins possessing ubiquitin-interacting motifs (UIMs): Eps15, Hrs and STAM2 On the basis of the above microscopical data, we conclude that the perinuclear clusters of APPL1 formed upon proteasomal inhibition co-localize with the commonly accepted aggresome markers.Since other endocytic proteins (i.e. Eps15) have been shown to affect aggresome formation The aggregates formed upon treatment with MG132 are reversible, and typically cells return to their normal morphology within 24–72 h after the removal of the inhibitor. To assess the effect of APPL1 on the clearance of aggresomes, after 18-h incubation with MG132 we washed the cells and allowed them to grow for the next 24 or 48 h in a normal medium. Upon 24 h of recovery time, ubiquitin was evenly distributed throughout the cytoplasm, with small cytoplasmic aggregates in 64% of the cells C. The agWe wanted to investigate whether relocalization of APPL1 to ubiquitin-rich aggresomes is related to any changes in its own ubiquitination status. To check if APPL1 is targeted by ubiquitin, we overexpressed APPL1 together with HA-ubiquitin in HEK293 cells. In such overexpression system we detected additional slower-migrating bands recognized by anti-APPL1 antibodies in APPL1 immunoprecipitates A. One stIn order to eliminate potential artifacts which could result from protein overexpression, we checked the ubiquitination of endogenous APPL1 in HeLa cells. We failed to detect ubiquitinated APPL1 under control conditions, but we could see polyubiquitinated species of APPL1 upon overnight treatment with MG132 B. Again,K63-linked polyubiquitination has been reported to promote sequestration of misfolded proteins into aggresomes and their subsequent clearance by autophagy One of E3 ubiquitin ligases participating in the modification of endocytic targets via K63-linked ubiquitin chains is Nedd4  As aggresomes contain high amounts of detergent-insoluble proteins In order to check whether ubiquitin conjugates could correspond to the observed additional bands of APPL1 appearing in an insoluble fraction of HeLa upon prolonged MG132 treatment A, we perEfficient protein biosynthesis is prerequisite for the formation of aggresomes stimulated by bortezomib Since impairment of proteasome machinery results in an accumulation of proteasomally degraded proteins, we checked for total levels of APPL1 in cells treated with MG132 with or without cycloheximide. HeLa cells treated for 20 h with DMSO or MG132 were incubated for the last 3 h or for the whole 20 h with cycloheximide. The normalized amounts of lysates were blotted for APPL1, Nedd4, EEA1, and control proteins: HDAC2 and α-tubulin C. CyclohRecent years brought a number of reports that describe the participation of an adaptor protein APPL1 in processes related to cellular growth, proliferation and survival, which at the molecular level are linked with signal transduction, membrane traffic, and gene transcription Given the growing importance of APPL endosomes, we wished to characterize their fate upon proteasomal stress. Strikingly, we observed a solubilization of APPL1 from endosomal membranes which made further observations of this endosome class impossible, since APPL1 is its unique marker. Instead, a prolonged treatment with all tested proteasome inhibitors caused clustering of APPL1 protein in the perinuclear region. Employing the commonly accepted criteria, we confirmed that endogenous APPL1 is recruited to aggresomes formed upon proteasomal inhibition. This discovery is particularly significant as there are only a handful of known endogenous proteins that become sequestered in aggresomes.It could be expected that the formation of an aggresome interferes with the proper localization of perinuclear cellular structures, such as the Golgi complex, mitochondria and lysosomes, which was confirmed in our observations. However, APPL1 endosomes are preferentially located at the cell periphery, and proteasome inhibition seems to affect specifically their marker protein APPL1 but not a marker of the canonical early endosomes EEA1. MG132 markedly decreases APPL1 labeling of endosomes, while the total amount of APPL1 protein in the cell is only slightly changed. The perinuclear APPL1 clusters form in about 50% of cells treated with MG132 for 20 h, and they meet the criteria describing aggresome formation: (a) the perinuclear clusters of APPL1 are surrounded by a cage of vimentin A; (b) thThere could be at least three explanations for the observed recruitment of APPL1 to aggresomes induced by the proteasomal stress. Firstly, if APPL1 was normally a target for proteasomal degradation, the blockage of proteasomes might have caused an excessive accumulation and aggregation of APPL1. Since we do not observe an increase in the total APPL1 protein level upon MG132 treatment, this simple explanation is unlikely. Secondly, proteasomal stress could cause damage to APPL1 which stays misfolded and aggregated due to the sequestration of cellular chaperones that could otherwise help to refold it. Thirdly, APPL1 might co-assembly with other aggregation-prone proteins. Since APPL1 co-localizes with aggregates formed by overexpressed GFP-Eps15 upon MG132 treatment D, one suIt is currently believed that aggresomes represent a protective cellular response to stress conditions. Importantly, the ubiquitin–proteasome system and protein aggregation are implicated in the pathogenesis of several genetic and neurodegenerative disorders The results of morphometric image analysis of APPL1- and EEA1-positive endosomes performed by MotionTracking. The table shows the raw data and the values calculated as a percentage of control values obtained from DMSO-treated cells. AU, arbitrary units; SD, standard deviation.The following are the supplementary materials related to this article.I.P. designed and performed the experiments and wrote the paper, L.S. analyzed the microscopical data with MotionTracking, Y.K. constructed the MotionTracking software and provided training of its usage, and M.M. provided conceptual advice and support and edited the manuscript.


The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin’s action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect.  Obesity increases the occurrence of several diseases and is a leading contributor to morbidity and mortality both in developed and developing countries. It is mainly caused by excessive food and energy intake .Since its discovery in 1994, leptin has been recognized as a hormone playing an important role in energy homeostasis . StudiesSimilar to other hormones, leptin exerts its effects by interacting with its receptors (OB-Rs) which are expressed in several peripheral tissues -16 inclu The secretion of leptin by the stomach and its entry with chyme into the small intestine suggest that it may play a role in food absorption. Indeed, several studies have shown that leptin interferes with the absorption of some nutrients. It enhances butyrate uptake , intestiLiterature on this topic is scarce  and the  The aim of this study was to investigate the direct effect of luminal leptin on glucose absorption by fully differentiated Caco-2 cells and to elucidate its signaling pathway, which although highly studied in the central nervous system, is still ill-defined in the peripheral tissues. The results might help in the design of new drugs that imitate leptin’s action and contribute to the treatment of obesity and other related disorders.-1 Glucose and pyridoxine HCL, Fetal Bovine Serum (FBS), Penicillin/Streptomycin (PS), Trypsin-EDTA, 10x Phosphate Buffered Saline (PBS) without calcium and magnesium were purchased from Sigma, Chemical CO, St. Louis, Missouri, USA. Phorbol-12-myristate-13-acetate (PMA), an activator of PKC, was purchased from CALBIOCHEM, San Diego, California, USA. Wortmannin, PD98059, SB202190, and Calphostin C, respective inhibitors of PI3K, MEK/ERK, p38MAPK and PKC, were also purchased from CALBIOCHEM, San Diego, California, USA. Transwell permeable supports  were purchased from Corning, MA, U.S.A. Scintiverse BD Cocktail was purchased from Fisher Scientific, Fairlawn, NJ, USA 3-O-methyl-D-glucose ([14C] 3OMG) and Mannitol, D-[1-3H (N)] were bought from Amersham International Ltd. Amersham, UK. The human colon carcinoma cell line (Caco-2) from a Caucasian male was purchased from American Type Culture Collection (ATCC), VA, USA. Human leptin was purchased from Biovision, CA, USA. Rabbit anti-ERK1/2 polyclonal antibody was purchased from Promega, WI, USA. Rabbit anti-p-p44/42 MAPK (ERK1/2) monoclonal antibody was purchased from Cell Signaling, MA, USA. Rabbit anti-Akt1/2/3, anti-p- Akt1/2/3, anti-p38α, anti-p-p38α polyclonal antibodies, and anti-rabbit IgG horse raddish peroxidase (HRP) conjugated were purchased from Santa Cruz, CA, USA. Protease inhibitors cocktail tablets were purchased from Boehringer Mannheim, Germany. Enhanced Chemiluminescence (ECL) kit was obtained from Santa Cruz, CA, USA. Nitrocellulose membranes, Biorad protein assay reagent and rainbow marker were purchased from Biorad, California, USA. Dulbecco’s Minimal Essential Medium (DMEM) with 4500 mg L-1 Glucose, sodium pyruvate, 1% Penicillin (100 µg mL-1), streptomycin (100 µg mL-1), 10% FBS, in a humidified incubator  at 37°C. The cells were grown either on 100 mm culture plates or on polyester transwell permeable supports  at a density of 120,000 cells mL-1 and treated always on the twenty third day after confluence following an overnight starvation.  Caco-2 cells were used at passages 25-35. They were grown in DMEM containing 4500 mg L14C methyl D glucose ([14C] 3OMG)  and D-mannitol-[1-3H (N)]  were added to the upper well of the transwell inserts on which Caco-2 cells were cultured. Mannitol was added to check for the integrity of the Caco-2 cells monolayer and to confirm junctional tightness. Samples were collected from the lower chamber immediately upon addition of the radioactive tracers and every 10 min thereafter, over a period of 50 min, and were assayed for radioactivity.  Leptin (10 nM), 3-O-The involvement of MEK/ERK, p38MAPK, PI3K, and PKC in the pathway mediating the effect of mucosal leptin on glucose absorption was studied. Starved fully differentiated CaCo-2 cells grown on transwell inserts for 23 days after confluence were pre-treated with a specific inhibitor of each of the above mediators and which are respectively: PD98059 (50 µM dissolved in DMSO), SB202190 (50 µM dissolved in DMSO), wortmannin (100 nM dissolved in DMSO) and Calphostin C (50 nM dissolved in DMSO). All inhibitors were added 15 min before leptin, except wortmannin and Calphostin C which were added respectively 20 min and 60 min prior to leptin. The vehicles were added to the control plates in the same amount and for the same duration.  In another set of experiments, the cells were also treated at the same dose and times described above but the four mediators were inhibited simultaneously in different combinations . In addition, PMA (100 nM dissolved in DMSO), an activator of PKC was also administered either alone or in the individual presence of inhibitors of MEK/ERK (PD98059), or p38MAPK (SB202190) or PI3K (wortmannin).  On day 23 after confluence, starved fully differentiated CaCo-2 cells grown on 100 mm culture plates were pre-treated, before leptin , with a specific inhibitor of each of the above mediators and which are: PD98059, SB202190, wortmannin and Calphostin C. The treatments were done at the same doses and times as described in part c above. At the end of the incubation period, the plates were washed twice with PBS buffer (pH 7.4) and the cells lysed and homogenized in a polytron  at 4°C after addition of a cocktail of phosphatase inhibitors. Proteins were quantified using the Bio-Rad assay. Equal amounts of proteins were loaded, resolved on 8% (Akt) or 10%  SDS polyacrylamide gel and transferred to a nitrocellulose membrane which was then blocked and incubated with a primary p38α, phospho-p38α, Akt1/2/3, phospho-Akt1/2/3, ERK1/2, or phospho-p44/42 MAPK (ERK1/2) antibody followed by an incubation with a goat anti-rabbit secondary horseradish peroxidase (HRP) conjugated IgG. The signal was detected by enhanced chemiluminescence using luminol. Equal loading was checked by GAPDH expression.  Results are reported as means ± SEM and tested for statistical significance by a one-way Analysis of Variance (ANOVA) followed by Tukey-Kramer multiple comparisons test using Instat and Excel Analysis softwares. Mucosal leptin (10 nM) caused a significant reduction in glucose absorption by fully differentiated CaCo-2 cells (23 days after confluence). This decrease appeared at 10 min and was maintained up to 50 min . -10 cm s-1 and 9.5 x 10-8 cm s-1. Monolayers in which the coefficient was higher than 5 x 10-7 cm s-1 were considered leaky and were discarded [ The apparent permeability coefficient of mannitol ranged between 6.6 x 10iscarded .  When Caco-2 cells were pretreated with PD98059, SB202190, wortmannin, or calphostin C, respective inhibitors of MEK/ERK, p38MAP kinase, PI3-kinase and PKC, the inhibitory effect of leptin on glucose absorption disappeared completely and went back to control values .  The effect of leptin on glucose absorption was similarly abrogated when Caco-2 cells were pre-treated simultaneously with calphostin C and SB202190, or calphostin C and wortmannin. Each combination of inhibitors alone did not have any effect on glucose absorption . Leptin could not exert its effect on glucose absorption in the simultaneous inhibition of MEK/ERK and PI3K, or the simultaneous inhibition of MEK/ERK, p38MAPK and PI3K .PMA, an activator of PKC, did not have any effect on glucose absorption . HoweverLeptin increased the phosphorylation of ERK (p-ERK), Akt (p-Akt) and p38MAPK (p-p38MAPK) .This increase changed into a decrease in presence of SB202190, an inhibitor of p38MAPK .The leptin-induced increase in the phosphorylated forms of Akt and p38MAPK still appeared when MEK/ERK was inhibited . No p-ERWhen PI3K was inhibited with wortmannin, leptin caused instead of an increase, a decrease in the level of p-ERK  and p-AkThis work demonstrated a significant inhibitory effect of mucosal leptin on glucose absorption in fully differentiated Caco-2 cells. Because extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), phosphatidylinositol-3-OH kinase (PI3K) and protein kinase C (PKC) are known to be involved in the regulation of glucose transport -36 , we  The results imply also that all four kinases involved in leptin’s action are along the same pathway, or else, a partial effect would have remained when every mediator was inhibited individually. To confirm that the four mediators act along the same pathway, they were inhibited simultaneously in different combinations. Leptin could not exert its effect when PKC was inhibited simultaneously with p38MAPK or simultaneously with PI3K . Thus, aAs the above results indicate that leptin acts via activation of PKC, we attempted to imitate the effect of leptin by activating PKC with PMA. To our surprise, PMA did not have any effect on glucose absorption, but caused a significant increase when administered simultaneously with PD98059, an inhibitor of ERK . These fSince all four mediators are along the same pathway, the positioning of one mediator with respect to the other needs to be determined. The activation of each mediator was checked by western blot analysis by determining changes in the level of their phosphorylated active forms or in the phosphorylation of their substrates. Leptin increased the phosphorylation of ERK  thus actTo position PI3K, the effect of leptin on the level of the phosphorylated form of its downstream target, the serine/threonine kinase Akt/PKB, was studied in presence of inhibitors of the different mediators. Akt is activated by phosphorylation on two residues, Thr308 and Ser473. An antibody directed against these residues was used. The level of p-Akt was increased by leptin . HoweverSince ERK was found to be the most downstream effector and to be directly activated by PI3K which is in turn activated by both PKC and p38MAPK, the positioning of p38MAPK and PKC with respect to each other still needs to be investigated. Leptin increased the phosphorylation of p38MAPK . This inIt can be concluded that leptin inhibits glucose absorption by activating PKC which acts via a stimulatory and inhibitory pathway. Leptin inhibits at the same time the stimulatory pathway and leaves only the inhibitory one active resulting thus in a reduction in glucose absorption. This inhibitory pathway involves activation of PKC which activates p38MAPK. The latter activates PI3K which eventually activates ERK. An active ERK exerts an inhibitory effect on glucose absorption. This work determined for the first time the direct effect and signaling pathway of mucosal leptin on glucose absorption by differentiated Caco-2 cells grown on transwell inserts. The identity of the mediator X needs still to be determined in a future work. These results suggest that leptin may induce weight loss not only by reducing appetite, but also by interfering with nutrient absorption. The findings provide experimental evidence supporting the benefit of leptin as a complementary therapeutic agent in the treatment of obesity and other related disorders.


Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both  These recent studies show that another mechanism whereby Src may promote cancer growth is by impeding the function of tumor suppressors, either causing the degradation or mislocalization of certain tumor suppressors. In this study, we asked whether Src could also regulate the ING1b tumor suppressor. The results demonstrate that Src is able to both physically associate with, and to phosphorylate ING1b. We found that Src decreases the stability of ING1b, and promotes ING1b relocalization from the nucleus to cytoplasm. In addition, we found that Src could functionally antagonize the ability of ING1b to induce apoptosis, suggesting that Src may promote tumour survival by reducing ING1 levels and causing ING1 relocalization.To ask if ING1 might serve as a substrate for Src, we performed immunoprecipitation-western assays to determine whether or not Src could physically interact with ING1. As shown in rd panel, lane1), and also in A431 cells  antibody , or kinase-dead Src (K298M). Cell extracts were immunoprecipitated with α-ING1 antibody, and blotted with α-pY antibody. As shown in in vivo. A decrease in total ING1 and in ING1 recovered in Src immunoprecipitates was also observed in cells expressing elevated levels of Src  was used to block protein synthesis, and ING1 levels were analyzed at 0 time and after 8 hrs of CHX treatment by western blotting. In the absence of elevated Src 80% of ING1 protein remained at 8hrs  by which ING1 stabililty is regulated appear to be complex, the level of this protein in cells is likely to have significant impact since it acts as a stoichiometric member of major histone deacetylase (HDAC) complexes Together with our previous report regarding Src-regulated degradation of the von Hippel-Lindau (VHL) protein that functions to inhibit vascular endothelial growth factor expression 2. Src plasmids used in our study, pCI-SRC, pCI-SRC Y530F, and pCI-SRC K298M, were generated using QuickChange® site-directed mutagenesis  and have been described previously HEK293, MDA-MB-468, and A431 cell lines were purchased from American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and grown at 37°C with 5% CO2, 150 mM NaCl, 1 mM DTT, 1 mM ATP) plus inhibitors  at 30°C for 30 minutes. The reactions were stopped with the addition of Laemmli sample buffer before being subjected to gel electrophoresis and western blotting.ING1 and Src proteins were purified as described Cell extracts in NP40 lysis buffer  supplemented with protease inhibitors  were immunoprecipitated with α-Src or α-ING1 antibodies for 2 hours at 4°C and then incubated for 1 hour at 4°C with a mixture of protein A and protein G beads. After immunoprecipitation, protein beads were washed four times with NP40 buffer and resuspended in sample buffer before gel electrophoresis and western blotting. BSA was used as a non-specific binding blocking agent, except for results shown in Cells were transfected as described above. Twenty-four hours post-transfection, cells were incubated with 100 µM cycloheximide (Sigma) for 0 and 8 hours before cell lysis and western blotting. The protein bands were scanned and quantified with a STORM 860 PhosphoImager .2, 200 µM sodium vanadate, 4 mg/ml p-nitrophenolphosphate). 50 µl of kinase assay buffer  and 100 µM Src optimal peptide was added to the immunoprecipitates and incubated for 15 minutes at 30°C. The reaction was stopped with 25 µl of 50% (v/v) acetic acid, after which 50 µl of the reaction mix was spotted on to a square of p81 phosphocellulose paper. The filter papers were then washed 5 times with 0.425% phosphoric acid, rinsed once with acetone and air dried before scintillation counting. To determine the specific activity of Src, a western blot of Src was performed on a duplicate set of Src immunoprecipitates from the same lysates, and the resulting bands were scanned and quantified with a STORM 860 PhosphoImager. The specific activity of Src (counts incorporated/band intensity) of each cell line represented is relative to the specific activity for SK-BR-3.Cells were lysed in RIPA buffer  supplemented with phosphatase and protease inhibitors . c-Src was immunoprecipitated from the cell extracts with 327 anti-Src antibody (1 µg of antibody per 100 µg cell extract) for 1 hour at 4°C followed by incubation with 40 µl of protein A and protein G bead mix (1∶1 ratio) for 1 hour at 4°C. The beads were then washed 4 times with RIPA buffer and once with Src dilution buffer  KinasePhos (bold) and Motif Scan (underlined) programs. A) Residues marked by asterisks have been previously reported to be phosphorylated. B) Tyrosine residues, predicted catalytic kinases and probability of the site being phosphorylated by the kinase using hidden Markov models . Tyrosines 55 and 212 show the highest probability scores for being direct Src substrates although there is variability between program predictions.(TIF)Click here for additional data file.