Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process. Lipid droplets are cellular organelles consisting of a neutral lipid core of triacylglycerides (TAGs), surrounded by a phospholipid membrane and a suite of proteins, which regulate lipid metabolism While it is clear that lipolysis is orchestrated by phosphorylation of several proteins, the specific relationship between phosphorylation events is not well understood. Murine perilipin 1A  reference sequence NP_783571.2) has six potential PKA phosphorylation sites HSL is phosphorylated on serines 563, 659, and 660 by PKA at the initiation of lipolysis. Phosphorylation of HSL on serines 659 and 660 are activating, but the function of phosphorylation on serine 563 is unclear The goal of the present study was to examine the specificity and time course of perilipin 1A phosphorylation at the initiation of lipolysis. Towards this end, monoclonal antibodies were developed which selectively label perilipin 1A phosphorylation at either PKA-site 5 (murine serine 492/human serine 497) or PKA-site 6 (murine serine 517/human serine 522). We utilized these reagents, in combination with high content analysis, to quantify the appearance of phospho-perilipin and phospho-HSL in adipocytes following exposure to lipolytic agents. We used FSK and isoproterenol, which increase cAMP, and L-γ-MSH, a peptide hormone that induces lipolysis by an incompletely characterized mechanism For certain experiments, human cells were utilized, which were obtained and de-identified by Zen-Bio, Inc, following written informed consent from the donors, utilizing a protocol approved by the Essex Institutional Review Board, Inc of Lebanon, New Jersey .2 flasks and allowed to grow to confluence. The cells were then exposed to Differentiation Media , 0.25 µM dexamethasone, and 1 µg/ml insulin). After 3 or 4 days in Differentiation Medium, the cells were harvested, seeded in 96-well glass bottomed dishes  precoated with gelatin which was cross-linked with glutaraldehyde 3T3L1 cells were obtained from ATCC and maintained in Growth Medium . Cells were passed a maximum of 17 times prior to use in experiments. To initiate adipogenesis, 3T3L1 cells were seeded in 75 cmHuman subcutaneous preadipocytes  were obtained via liposuction by Zen-Bio  and were plated  on glass-bottomed 96-well dishes precoated with gelatin that was cross linked with glutaraldehyde. To induce differentiation of the cells to adipocytes, the cells were exposed to Differentiation Medium , which contains factors that induce adipogenesis in this cell type; alternatively, in certain experiments, the cells were exposed to a similar media, which featured the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone (300 nM), which is also effective at inducing differentiation.The cDNA sequence encoding human perilipin 1A was purchased from Origene . S497A and S522A mutations were introduced by using internal primers including the sequence encoding each serine. The codon for serine was altered to encode alanine. Subsequently, the wild-type or mutated perilipin 1A cDNAs were transferred to either GFP or mCherry vectors using appropriate restriction sites. Wild-type and mutated forms of perilipin 1A were sequenced to verify fidelity. Antibodies used to visualize HSL or perilipin 1A were from Vala Sciences Inc . GP29, a guinea pig polyclonal antibody raised against perilipin, was from Progen . 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene  was used to visualize lipid droplets. Lipofectamine 2000, AlexaFluor 647-anti mouse secondary antibody, Cell Mask Blue, HCS LipidTox DeepRed, and SlowFade Gold mounting solution were from Life Technologies . HiLyte Fluor™ 647 goat-anti-rabbit (far-red fluorescent secondary antibody) was from AnaSpec . Texas Red conjugated goat-anti-mouse (red fluorescent secondary antibody) was from Jackson ImmunoResearch Laboratories.Mouse monoclonal antibodies were developed and characterized by the Monoclonal Antibody Core Facility at Baylor College of Medicine. Mice were inoculated with peptides containing amino acid sequences identical to those surrounding PKA-site 5 or PKA-site 6 of the human perilipin 1A sequence . N-α-FmoPLIN1 (wild type), mCh-PLIN 1 S497A, or mCh-PLIN1 S522A) used per transfection reaction. The next day, the cells were detached and seeded on 22 mm poly-D-lysine coated coverslips placed in 24-well dishes. Approximately 8 hr later, oleic acid complexed to fatty acid-free BSA  was added to induce lipid droplet formation. After an overnight incubation with oleic acid, the cells were treated with forskolin (24 µM) for 7 min to increase PKA activity. Treatments and transfections were performed in OPTI-MEM (Invitrogen).After isolation of hybridomas secreting monoclonal antibodies, specificity to codon mutation was tested using wild-type and mutant perilipin constructs in GFP and mCherry vectors, respectively. HeLa cells were plated in 6 well plates 24 h prior to transfection. HeLa cells were transfected using Lipofectamine 2000 with a single plasmid  for 30 minutes at room temperature. Coverslips were quenched with 100 mM ammonium chloride and washed three times with PIPES/HEPES/EGTA/MgCl2 (PEM) buffer, prepared at a final pH of 6.8. Cells were permeabilized with 0.2% Triton X-100 in PEM for 10 minutes, washed three times with PEM and blocked in 5% milk in PEM/0.01% saponin blocking solution for 30 minutes at room temperature. Antibodies were then diluted in the blocking solution and incubated overnight at 4°C. Subsequently, coverslips were washed with PEM and incubated with AlexaFluor 647-conjugated anti-mouse secondary antibodies (Life Technologies) for 1 h at room temperature. Cells were again washed 3 times and incubated with CellMask Blue (1 µg/ml), and DAPI (10 µg/ml) in PEM for 45 minutes at room temperature. Dyes were then aspirated, PBS/0.01% azide added, and cells imaged immediately. Coverslips were mounted with SlowFade Gold (Invitrogen). Images were acquired with a Beckman coulter IC-100 Image Cytometer equipped with a Nikon S Fluor 20X/0.75NA objective or a DeltaVision Core Image Restoration Microscope  using a 60X, 1.42 NA Plan Achromat objective , and a Photometrics CoolSnap HQ2 CCD camera.Preadipocytes were obtained from Zen Bio  and were plated directly on 96 well plate at the density of 15000 cells per well. siRNA transfection was performed at the time of plating. A mix of the cells, media , siRNA , and transfection reagent  was prepared, and 200 µl of the mixture was added per well. The sense sequence of the perilipin 1A siRNA was GCACAUACCCUGCAGAAGA (5′ to 3′); additionally, an siRNA that does not correspond to a human gene was utilized as a control. The perilipin 1A and control siRNAs were tested at four different concentrations: 0 nM, 10 nM, 20 nM, and 50 nM. 48 hours post transfection, transfection media was removed, and differentiation medium was added for 6 days. Cells were then treated with forskolin at a final concentration of 6 µM in DMEM for 30 minutes, then fixed and labeled for nuclei, lipid droplets, and primary antibodies  via methods described below.Cells were plated on 96-well dishes and exposed to experimental treatments, after which they were fixed with 4% paraformaldehyde plus 0.025% glutaraldehyde. The cells were then permeabilized with 0.1% Triton-X-100 (20 minutes) and blocked for 60 minutes in blocking buffer . Fixed cells were labeled with primary antibodies diluted in blocking buffer for either 60 minutes at 37 C with rotation, or overnight at 4 C. The samples were then rinsed 3X with PBS, incubated for one hour with appropriate secondary antibodies  conjugated to red channel or far-red channel fluorophores, along with the Lipid Droplet Stain to label lipid droplets in the green channel. Following incubation with the secondary reagents, the plates were rinsed 3X with PBS. DAPI was then added; the dishes then sealed and incubated a further 20 minutes at room temperature, prior to automated image acquisition. In experiments in which peptides were tested for their ability to block primary antibody binding, the primary antibodies were preincubated with 0 to 10 µg peptide in a volume of 300 µl for 30 minutes at 37 C.Images were acquired with a Beckman Coulter IC 100 using established methods An LSM 710 NLO Zeiss Multiphoton Laser Point Scanning Confocal Microscope was used to scan adipocytes labeled with newly developed phospho-perilipin 1A specific antibodies, and a reference antibody to perilipin 1A. For these scans, a multi-photon Mai-Tai DeepSee HP laser was used to excite DAPI with 728 nm (pinhole = 601 µm); single channel lasers at 514 (31 µm), 561 (36 µm), and 622 (42 µm) were used for image acquisitions in the green, red, and far-red fluorescence channels, respectively. 20×0.8 NA M27 Plan apochromat and 63×1.4 NA M27 DIC objectives were used.2/cell). A second data parameter, Tii Pi Pm, is reported for certain experiments in which the effect of blocking peptides were tested for their ability to reduce the labeling intensity obtained with the primary antibodies. Tii Pi Pm is the “Total integrated intensity” (sum of intensities of the pixels), in the “Protein image” (the image channel representing either perilipin 1A or HSL), in the “Protein mask” . Thus, Tii Pi Pm reports the overall intensity of the cell for the protein that is labeled CyteSeer®  is a Java-based PC/Mac/Linux- compatible cell image analysis program designed specifically for high content analysis. CyteSeer®’s Colocalization Algorithm has been specifically developed for the analysis of lipid droplets and lipid droplet-associated proteins 3T3L1 cells were plated and differentiated in 6 well plates. After stimulation, cells in each well were rinsed with ice-cold PBS and lysed in 75 µl buffer containing 50 mM Tris, pH 7.4, 100 mM NaCl, 1% sodium deoxycholate, 4% Nonidet P-40, 0.4% SDS, proteases and phosphatase inhibitors (Sigma). Lysates were incubated on ice for 15 min, frozen overnight at −80°C, thawed on ice and centrifuged at 13,000 rpm. The resulting supernatants were stored at –80°C, and protein concentrations were determined using the BCA kit . Next, 5–40 µg of lysate were loaded on 12% BisTris gel, subjected to electrophoresis and transferred to BiotraceTM PVDF membrane . After blocking with 5% milk protein in TBST for 1 hour, membranes were incubated overnight with primary antibodies. Primary antibodies were detected using an HRP-conjugated secondary antibody and visualized by ECL.GraphPad Prizm was used to test for statistically significant differences between controls and experimental groups .To generate novel monoclonal antibodies directed to phosphorylated forms of perilipin 1A, peptides analogous to the amino acid sequences surrounding serine 497 (PKA-site 5) or serine 522 (PKA-site 6) of human perilipin 1A were synthesized. The serines of these peptides were substituted with a chemical moiety , which acts as a mimetic for phospho-serine. Mice were injected with the peptides, and sera-positive animals were screened for MAb-secreting hybridomas. Aliquots of media from the hybridomas were tested against the inoculating peptides via ELISA. Clones that scored positive for recognition of the inoculating peptides by ELISA were evaluated for their ability to produce antibodies that visually label adipocytes pretreated with forskolin in a pattern consistent with perilipin 1A. We selected two monoclonal antibodies, designated anti-pPeri-site 5 and anti-pPeri-site 6, that showed strong ELISA reactions and appropriate labeling patterns.To evaluate the specificity of the anti-pPeri-site 5 and anti-pPeri-site 6 antibodies, perilipin 1A mutant plasmids were utilized, in which alanine was substituted for either serine 497 (to remove PKA-site 5) or serine 522 (to remove PKA-site 6). The coding region of each perilipin 1A mutant construct was fused to mCherry, a red channel fluorescent protein, to create mCh-perilipin S497A and mCh-perilipin S522A. Separate HeLa cell preparations were transiently transfected with GFP-perilipin 1A (wt), mCh-perilipin 1A (S497A), or mCh-perilipin 1A (S522A). The cells were then exposed to oleic acid to promote formation of lipid droplets for 24 h. Subsequently, FSK+IBMX was used to activate PKA. Cells were then fixed, stained for nuclei, and labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. Antibody labeling was visualized with secondary anti-mouse antibodies conjugated to a far-red fluorophore.For cells prepared in this manner, the anti-pPeri-site 5 antibody labeled cells expressing wild type perilipin 1A , but didGuinea pig polyclonal antibody #GP29, raised against the N-terminus of perilipin 1A, is widely used to label human r has a theoretical range from −1.0 (perfect exclusion) to +1.0 (perfect coincidence). Thus, the observed Pr values strongly support the observation from visual inspection that distribution of phospho-perilipin 1A labeled by the monoclonal antibodies coincides with the distribution of total perilipin 1A labeled by GP29. For all of the antibodies, labeling was specific for the edges of lipid droplets, which is the known intracellular location of perilipin 1A. Virtually identical images for anti-pPeri-site 5 and GP29 and for anti-pPeri-site 6 and GP29 were also obtained by confocal microscopy  or an siRNA corresponding to human perilipin 1A, at concentrations ranging from 0 to 50 nM. Following transfection, the cells were exposed to Differentiation Medium for 6 days, treated with 6 µM FSK for 20 minutes, and fixed and labeled for nuclei, lipid droplets, and for both phospho-perilipin (utilizing either anti-pPeri-site 5 or anti-pPeri-site 6) and total perilipin (utilizing GP29). For cells transfected with 10 nM control siRNA, most of the cells featured numerous lipid droplets and were strongly labeled by GP29, anti-pPeri-site 5, and anti-pPeri-site 6 . In contTo quantify the effects of the siRNAs, images from the experiment were analyzed with CyteSeer®. Area Pm values were not affected by the control siRNA, but the perilipin siRNA reduced labeling by GP29, anti-pPeri-site5, and anti-pPeri-site 6 in a very similar fashion, up to 90% .The above results confirm that anti-pPeri-site 5 labels perilipin 1A which has been phosphorylated at PKA-site 5, whereas anti-pPeri-site 6 labels perilipin 1A, which has been phosphorylated at PKA-site 6. Note that for the purposes of quantification, perilipin 1A labeled by the anti-phospho-perilipin 1A antibodies, will be referred to as pPeri-site 5 and pPeri-site 6, respectively.Next, experiments were conducted to test the ability of these antibodies to recognize endogenous phospho-perilipin 1A in adipocytes under lipolytic treatments. Human adipocytes exposed to control medium showed little labeling for with the anti-pPeri-site 5 antibody . In contWe 2/cell for control cells  averaged 27 µmol cells , suggest  . Thus, bIn a reciprocal experiment, also featuring 3T3L1 adipocytes treated with 1 µM isoproterenol, preincubation of anti-pPeri-site 5 antibody with the pHSLser660 peptide did not reduce overall image intensities or Tii Pi Pm obtained with the anti-pPeri-site 5 antibody ; in contFinally, for 3T3L1 adipocytes treated with 100 nM L-γ-MSH, preincubation of anti-pPeri-site 6 antibody with a peptide corresponding to pPeri-site 6 strongly reduced image intensities and the Tii Pi Pm values obtained with anti-pPeri-site 6 antibody, but did not reduce overall image intensities or the Tii Pi Pm values obtained with the anti-pHSL-serine 563 antibody . The resOverall, the results with the blocking peptides confirmed the specificity of the phospho-perilipin and phospho-HSL antibodies for labeling their intended targets and indicate it is highly unlikely that the anti-perilipin 1A antibodies recognize HSL, or vice versa.To explore additional applications for the monoclonal antibodies against phospho-perilipin, we tested the ability of these antibodies to identify phospho-perilipin 1A by Western blotting. In our hands, anti-pPeri-site 5 did not visualize specific bands on Western blots. In contrast, anti-pPeri-site 6 labeled a single band, which migrated at approx. 60 KDa, which is consistent with the molecular weight of perilipin 1A, and the intensity of this band was strongly increased in samples for 3T3L1 cells treated with forskolin . FurtherThe goal of this study was to examine the phosphorylation of perilipin 1A for comparison to HSL phosphorylation, at the initiation of lipolysis. Towards this purpose, we developed novel monoclonal antibodies (anti-pPeri-site 5 and anti-pPeri-site 6), which specifically recognize perilipin 1A phosphorylated at either PKA site 5 or PKA site 6, respectively. The specificity of these antibodies was confirmed in several ways. First, these antibodies recognized the synthetic phospho-peptides corresponding to the perilipin 1A PKA phosphorylation sites used in immunizing the mice, by ELISA. In subsequent experiments, these antibodies labeled ectoptically expressed perilipin 1A in FSK/IBMX-treated HeLa cells with no labeling of other proteins endogenous to these cells. Also, the labeling of ectopic perilipin 1A in HeLa cells was eliminated by single point mutations in perilipin where alanine was substituted for serine at the target site. The labeling by each antibody was also increased by FSK, an agent known to stimulate PKA-mediated phosphorylation of perilipin. The anti-pPeri-site 5 and anti-pPeri-site 6 antibodies labeled the edges of lipid droplets, consistent with the cellular localization pattern for perilipin 1A, and the labeling patterns were coincident with labeling by GP29, a well-established guinea pig polyclonal antibody to perilipin 1A. Finally, for FSK-treated human adipocytes, an siRNA specific for perilipin 1A down-regulated labeling by anti-pPeri-site 5, anti-pPeri-site 6, and GP29 in an identical manner. As pPeri-site 5 and pPeri-site 6 are associated with the carboxyl terminal of the protein, the antibodies are specific perilipin 1A, which is the full-length form of perilipin Our results demonstrate that both perilipin 1A and HSL have sites that are phosphorylated at different rates in response to lipolytic agents and these sites are phosphorylated in response to lipolytic agents, in a strikingly similar manner. For example, perilipin 1A PKA-site 5 and HSL-serine 660 were phosphorylated rapidly and with equal efficacy in 3T3L1 adipocytes exposed to either FSK or L-γ-MSH. In contrast, perilipin 1A PKA-site 6 and HSL serine 563 were phosphorylated more slowly, and L-γ-MSH was a markedly stronger agonist for this effect than FSK. Indeed, when cells were exposed to an extended panel of lipolytic agents, the pattern of results for pPeri-site 5 was virtually identical to that for pHSL-serine 660, whereas the pattern of results for pPeri-site 6 were very similar to that for pHSL-serine 563. These similarities were not due to recognition of HSL by the antibodies raised to perilipin 1A or recognition of perilipin 1A by the antibodies raised to HSL, as the antibodies were selectively blocked by peptides that correspond to their intended targets.The differential phosphorylation of HSL (more rapid at serine 660 vs. serine 563 in response to FSK) was first observed by Martin and colleagues Consideration of the amino acid sequences of the PKA phosphorylation sites on perilipin 1A and HSL leads us to suggest an alternative hypothesis to account for the differential phosphorylation of these proteins. The consensus sequence for PKA is considered to be R(R/K)X(S/T) Indeed, perilipin 1A PKA-site 5 and site 6 have previously been linked to different physiological processes , might be a candidate for this as it shares substrate specificity with PKA and is present in 3T3L1 adipocytes High content analysis methods provide several advantages over traditional immunofluorescence labeling and analysis techniques. The high throughput plating configuration enables processing of a larger number of samples per experiment than is practical with procedures in which cells are plated on coverslips, which must be handled individually and mounted on slides. Secondly, the image analysis algorithms objectively analyze each cell imaged, providing quantitative data for hundreds of cells per condition. High content analysis permits discrimination between effects subtly different between experimental treatments and greater resolving power for statistical analysis. While high content analysis strategies have most commonly been utilized in high throughput screening applications, our study demonstrates the value of utilizing HCA to examine the early events associated with activation of lipolysis, a pathway of key importance to obesity and the metabolic syndrome. Our data suggests future directions for research such as testing the hypothesis that differential phosphorylation of perilipin 1A and HSL depends upon the amino acid sequences flanking the phosphorylation sites, and to pursue further elucidation of the mechanisms by which L-γ-MSH elicits phosphorylation of perilipin 1A and HSL.


Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations . Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation. The sequences were cloned into the pT-REx vector (Life Technologies) using standard cloning procedures. The monomeric Emerald fluorescent protein  and plated into tissue culture treated optical-grade 8-well chamber slides that were pre-coated with 0.01% (w/v) poly-L-lysine for 30 min, washed with water and dried for 2 h. The cells were incubated for 24 h expression. For Western blotting, 1×106 cells were plated into 25 cm2 tissue culture flasks. After 24 h cells were rinsed in serum reduced Opti-MEM and then transfected using 2 µg of DNA and 5 µL of Lipofectamine 2000. After 6 h transfection, the media was replaced with supplemented DMEM. For analytical ultracentrifugation, 4×106 cells in 75 cm2 tissue culture flasks were transfected with 24 μg DNA and 60 μL Lipofectamine 2000 as described for Western blotting, with the media replaced after 7 hours. The cells were incubated for 24 h expression.All experiments were performed in AD293 cells, which were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FCS, 2 mM glutamine, 200 U/mL penicillin and 200 µg/mL streptomycin. The culture flasks were kept at 37°C in a humidified incubator with 5% atmospheric COg for 3 min at room temperature. The pellet was resuspended in 5 mL ice-cold phosphate-buffered saline (PBS) by gently pipetting up and down. The cells were again pelleted by centrifugation at 3315×g for 3 min at 4 °C. The pelleted cells were lysed with 300 µL of RIPA buffer  Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.2% (w/v) sodium fluoride, 0.2% (w/v) sodium orthovanadate, 20 U/mL benzonase and Complete EDTA-free protease inhibitor (Roche Diagnostics)) on ice for 40 min. Aliquots were then snap-frozen in liquid nitrogen and stored at −80°C. Total cellular protein concentration was determined in triplicate using a BCA protein assay using bovine serum albumin as the standard, according to manufacturer's instructions (Thermo Scientific). Protein samples were resolved by 12% (v/v) polyacrylamide SDS-PAGE and transferred to PVDF membrane at 100 V for 1 h in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3, using a Criterion Blotter Transfer System (Bio Rad). The membrane was blocked with 5% (w/v) skim milk powder or 3% (w/v) BSA for the phospho-specific antibodies in PBS-T  Tween-20) for 1 h at room temperature or overnight at 4°C. Primary antibodies were added to the blocking buffer for 2 h at room temperature or overnight at 4°C. Antibodies were used at 1∶10,000 dilution for GFP , 1∶1,000 dilution for STAT3 , 1∶1,000 for STAT3 pY705 , 1∶500 c-Src , 1∶7,500 for c-Src pY416#1 , 1∶7,500 for c-Src pY416#2 , 1∶10,000 for α-tubulin (Invitrogen). The membrane was then washed four times over 30 min with PBS-T before probing with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membrane was washed four times over 30 min with PBS-T. The protein bands were visualized using enhanced chemiluminence according to manufacturer's instruction (Sigma-Aldrich) and digital acquisition by a LAS-3000 imager (Fujifilm).Cells were harvested in 5 mL DMEM with a cell scraper. The cell suspension was transferred into the 15 mL plastic tube and centrifuged at 3315×g; 5 min room temperature) and pellet was resuspended on ice in 1 mL ice cold native lysis buffer (NLB)  Triton X-100, Complete-EDTA-free protease inhibitor (Roche Diagnostics), 0.2% (w/v) sodium fluoride, 0.2% (w/v) sodium orthovanadate, 20 U/mL benzonase, 1 mM PMSF). Cells were mechanically lysed on ice by extrusion through an 27 Gauge needle 20 times. 5 M NaCl was added to a final concentration of 150 mM. Lysate was maintained on ice until analytical ultracentrifugation. Total protein concentration was measured using a BCA assay and adjusted to 0.5 mg/mL with NLB. Lysate was loaded into Charcoal-Epon 12 mm thick Velocity quartz-window centerpieces, prechilled at 4°C overnight, and centerpieces placed into a prechilled AnTi-8 rotor also prechilled at 4°C overnight. Samples were analyzed with an XL-A analytical ultracentrifuge (Beckman Coulter) equipped with a fluorescence detection module (AVIV) and 488 nm laser at 10°C. Continuous radial fluorescence scans were collected at a rotor speed of 3,000 rpm for 2 h. The rotor speed was increased to 40,000 rpm and a further 200 scans were collected. The sedimenting boundaries were fitted to a model describing the sedimentation of a distribution of sedimentation coefficients c(s), which were converted to mass units, c(M), using the program SEDFIT Transfected cells were harvested by scraping off the plate in 5 mL DMEM and placed into a tube. Cells were pelleted  We next examined the dependency of Y416 phosphorylation on expression level of c-Src. Various doses of constitutively open and closed c-Src variants were transfected into AD293 cells with DNA titrated against a plasmid expressing a non-fluorescent Y66L GFP derivative s20,w = 4.3 S and 6.5 S)    and 7. Tosition) . These dosition) . This reHere we show that as per previous studies, levels of Y416 phosphorylation is correlated to the open active conformation of c-Src Prior studies have suggested that Src family kinases have a more complex multi-state conformational regulation than a two-state “open” and “closed”. Specifically, it was reported that the constitutively closed YEEI Hck mutant could be activated by the high-affinity SH3 ligand Nef We propose that c-Src autophosphorylation may occur partly by the dimerization of c-Src, which we showed occurs for both open and closed c-Src, but which trends to higher amounts for the open state and at higher levels of expression. Hence, both open and closed c-Src participate in monomer-dimer equilibria that seems to influence Y416 autophosphorylation.The autophosphorylation of Y416 in the closed state in a concentration-dependent manner has intriguing implications for the mechanisms of c-Src regulation. These findings point to a model whereby Y416 autophosphorylation could act as a mechanism to prime locally concentrated closed c-Src for high activity upon kinase opening by secondary events. This mechanism of control of c-Src activity is conceivably important for rapidly modulating and amplifying c-Src activity at focal adhesions, where c-Src is locally enriched and engaged with focal adhesion kinase, which binds to c-Src through the SH2 domain in the open conformation The second implication is that because STAT3 phosphorylation is much more dependent on c-Src being in the open conformation than autophosphorylation, then priming of Y416 will not be sufficient to activate substrate phosphorylation. Previous studies have suggested that STAT3 phosphorylation requires c- Src to have a functional SH3 domain


Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin.2+ sensitivity.In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament CaApelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure. APLNR) Apelin is the endogenous ligand of the former ‘orphan’ APJ receptor, a G-protein-coupled receptor (GPCR), now known as the apelin receptor  and phenylephrine, occur mainly via myofilament Ca2+ sensitization +-H+ exchanger (NHE) with subsequent intracellular alkalization 2+ rather than increasing intracellular Ca2+ transients Apelin has been identified as one of the most potent endogenous stimulators of cardiac contractility All protocols were reviewed and approved by the Animal Use and Care Committee of the University of Oulu.Drugs used were Apelin-16 ; Bisindolylmaleimide I (Bis) ; ML-7 ; U0126 . Apelin was dissolved in 0.6% acetic acid; Bis, ML-7 and U0126 were dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration was <0.15% in the perfusion buffer. DMSO and acetic acid were added to each vehicle control experiments in volumes equal to those used for diluting the drugs in parallel experiments.2 and 5% CO2 at 37°C. Hearts were perfused at a constant flow rate of 5.5 mL/min with a peristaltic pump . Heart rate was maintained constant (305±1 beats per minute) by atrial pacing using a Grass stimulator  . Contractile force  was obtained by connecting a force displacement transducer  to the apex of the heart at an initial preload stretch of 20 mN. Perfusion pressure reflecting coronary vascular resistance was measured by a pressure transducer  situated on a side arm of the aortic cannula. Data were recorded using IX-228 Data Acquisition System and LabScribe recording and analysis software .Male 7-week-old Sprague-Dawley rats from the Center for Experimental Animals at the University of Oulu were used (n = 134). Rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique as described previously The experiments started with an equilibration period (40±4 min) and a 5-minute control period. Then various drugs were added to the perfusate for 5, 10, 15 or 20 minutes. Drugs used were apelin-16 (apelin); Bis; ML-7 and U0126. The doses of apelin were determined by pilots before each set of experiments to achieve comparable inotropic responses to that of our previous study We followed the protocol found to be the most effective in preserving PKC isoforms by Hunter and Korzick LV samples were subjected to urea/glycerol poliacrilamide gel electrophoresis (PAGE) to separate phosphorylated and nonphosphorylated RLC as described previously 3VO4, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 2 μg/mL pepstatin, 2 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride and 20 mmol/L NaF. Samples were then centrifuged at 1300×g for 5 min at 4°C and the supernatant was collected. Protein concentrations were determined by the method of Bradford. Protein extracts were matched for protein concentration and stored denaturated in SDS loading buffer at −70°C. Equal volumes (30 μg) of protein samples were loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes. Protein levels were detected using fluorescence as described above. Quantification of the blots was done by using the Quantity One Basic 1-D Analysis Software . The antibodies used were anti-phospho-ERK1/2, anti-ERK1/2, anti-p38, and anti-phospho-p38 .Frozen LV tissues were grinded in liquid nitrogen and were dissolved and homogenized in ice-cold lysis buffer containing of 20 mmol/L Tris, (pH 7.5), 10 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, supplemented with 1 mmol/L β-glycerophosphate, 2 mmol/L dithiothreitol, 1 mmol/L NaP<0.05.SPSS Statistics 20 program was used for statistical analysis. Results are presented as mean±SEM. Repeated-measures ANOVA test was used to evaluate the statistical significance of differences among groups for cardiac contractility. The two factors were treatment group  and time as the repeated measure. When significant differences were detected in 2-way repeated measures ANOVA for the treatment-by-time interactions, a Bonferroni post hoc test was used for specific comparisons. 2 groups at a time were compared using unpaired Student’s t test; all other parameters were analyzed with 1-way ANOVA followed by Bonferroni post hoc test. Differences were considered statistically significant at the level of P<0.001; 1]apelin-13, apelin-13 and apelin-36 have comparable potency and efficacy in inducing positive inotropic effect in human paced atrial strips In the isolated perfused rat heart preparation, administration of apelin (2 nmol/L) for 20 min induced a slowly developing and sustained increase in cardiac contractility . Infusion of ML-7 alone had no significant effect on contractile force when compared to vehicle control , with a maximum increase of 99±23% at 10 min  or in combination with apelin , the upstream regulator of ERK1/2. The inotropic effect of apelin was significantly attenuated by U0126 (5 μmol/L), the maximal reduction being 56% (P<0.05). Infusion of U0126 alone had no significant effect on contractile force P = 1.0; . ImmunobParticulate partitioning of PKCε in neonatal rat ventricular myocytes is accompanied by subsequent activation of ERK1/2 Apelin is among the most potent endogenous inotropes yet identified 2+ sensitivity of myofilaments http://www.kinase-screen.mrc.ac.uk/kinase-inhibitors). Therefore it is plausible to assume that the apelin-mediated increase in cardiac contractility is partly dependent on MLCK activation. Nevertheless, no significant apelin-induced increase in RLC phosphorylation was detected by urea-glycerol PAGE. One should consider, however, that given the rate of approximately 40% of RLC phosphorylation under basal physiological conditions ex vivo experimental conditions.RLC controls myofilament cross-bridge properties and thereby modulates the force of contractions in the heart. RLC is phosphorylated by the cardiac MLCK 2+/calmodulin-independent. On the other hand, potential phosphorylation sites for PKC were identified on cardiac MLCK The exact mechanisms of cardiac MLCK activation remain elusive. Contrasting smooth- and skeletal muscle isoforms, cardiac MLCK was found to be Ca2-adrenergic receptor or endothelin receptor stimulation increases p38-MAPK activation, and pharmacological inhibition of p38-MAPK activation augments β2-adrenergic receptor - or endothelin-mediated increases in cardiac contractility The MAPKs are well known regulators of diverse processes in the heart under physiological and pathophysiological conditions q-overexpressing mice. Reciprocally, enhanced PKCε activation resulted in a more favorable LV geometry with improved LV performance in Gαq mice, displaying concentric instead of eccentric remodeling Accumulating lines of evidence suggest that the activation of PKCε and MEK1/2–ERK1/2 cascades constitute important adaptive mechanisms in the myocardium under pathological conditions. PKCε and ERK1/2 signaling have been reported to confer cardioprotection in vivo against ischemia-reperfusion injury by reducing cell death 2+-sensitivity of the contractile machinery. Modest activation of PKCε and MEK1/2–ERK1/2 signaling have been reported to confer protection against stress-induced myocyte apoptosis The present study provides evidence that apelin increases cardiac contractility through parallel and independent activation of PKCε and MEK1/2–ERK1/2 signaling in the adult rat heart. Moreover, MLCK activation represents a downstream mechanism by which apelin may increase Ca


Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform . We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. Insulin resistance (IR) is defined as a state in which normal amounts of insulin produce a subnormal biological response Glucocorticoids (GCs) are well known to induce IR in a variety of clinical and experimental settings Tau hyperphosphorylation is one of the hallmark pathological alterations in AD Calpains, a family of calcium-activated cysteine proteases, have been reported to be involved in the pathogenesis of AD To address the issues mentioned above, we employed two cell lines, wild-type HEK293 cells HEK293/tau441 cells, a good cellular model to study tau pathology Mouse monoclonal antibody (mAb) against β-actin was purchased from Santa Cruz Biotechnology . Rabbit mAb against Akt and phosphorylated Akt at Ser473 (pAkt) were obtained from Cell Signaling Technology . Mouse mAb against μ-calpain was purchased from Sigma . Mouse mAb against Tau-1 was obtained from Millipore . The antibody R134d that recognizes total tau was generously provided by Gong Cheng-Xin . Dexamethasone (DEX), insulin, mifepristone (RU), lithium chloride (LiCl) and E-64d  were purchased from Sigma .HEK293 cells stably transfected with the longest isoform of recombinant human tau  and HEK293 cells transfected with an empty vector (HEK293/vec) were a generous gift from Professor Qing Tian . The construction of HEK293/tau441 and HEK293/vec cells is as previously described For the experiments, both HEK293/tau441 and wild-type HEK293 cells were first incubated in DMEM with 2% FBS overnight in order to reduce the serum effects. The cells were then pre-treated with RU , LiCl , or E-64d  and then treated with DEX (1 µM), or treated with DEX (1 µM) alone. For treatment longer than three days, the medium was changed at 72 hours (h) after exposure to DEX. Then RU (20 µM), LiCl (10 mM) or E-64d (30 µg/ml) with DEX (1 µM) or DEX (1 µM) were added to the media. After incubation with the above agents, cells were exposed to 100 nM insulin for 15 minutes (min) and then harvested for Western blotting.g for 15 min. The protein concentration in each lysate was determined using a bicinochonic acid (BCA) Protein Assay Kit  with the bovine serum albumin as the standard. Equal amounts of proteins (60–80 µg) were separated on 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in TTBS  and probed overnight at 4°C with antibodies against β-actin (1∶400), pAkt, Akt, Tau-1 and μ-calpain (1∶1000) and total tau . The blots were developed with horseradish peroxidase-conjugated secondary anti-mouse  or anti-rabbit antibodies  and visualized with an enhanced chemiluminescent substrate kit  and exposure to CL-XPosure film (Kodak). The films were scanned and the density of each of band was quantified using Kodak Digital Science 1D software .The total proteins from the cultures were extracted with the ice-cold buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 100 µM sodium orthovanadate, 1 mM phenylmethysulfonyl fluoride and 1 µg/ml protease inhibitor cocktail . The cell lysates were boiled, briefly sonicated and centrifuged at 12500×post hoc pair-wise comparisons. P<0.05 or lower was considered to represent statistically significant differences.Numerical data were expressed as group means ± SEM and analyzed with SPSS statistical software (version 13.0). All data sets were subjected to one-way ANOVA before application of Materials and Methods) to mimic the induction of long-term stress in vivoThe insulin-stimulated increase in pAkt is a key event in the proximal insulin signalling pathway and the pAkt/Akt ratio has been widely used as a marker for evaluating IR Materials and Methods) did not affect the pAkt levels upon DEX treatment  and the other is mineralcorticoid receptor (MR). Because HEK293 cells do not express MR  lane 3, . However lane 3, , indicatversus D6). Because GCs are known to induce tau phosphorylation Although DEX inhibited the insulin-stimulated Akt phosphorylation in wild-type HEK293 and HEK293/tau441 cells, the inhibitory effect of DEX occurred earlier in HEK293/tau441 cells  for 3 days (D3) or 6 days (D6). Total amounts of tau (detected by R134d) remained stable under the conditions and were used as internal standards for measurement of the relative level of Tau-1 , an inhibitor of glycogen synthase kinase-3β (GSK-3β) that has been shown to inhibit tau hyperphosphorylation in different contexts Materials and Methods) for 3 days (D3), followed by insulin stimulation. Total amounts of tau remained stable under the conditions  and the inactive/full-length calpain (80-kDa band). DEX did not affect μ-calpain activation in wild-type HEK293 cells on D3 or on D6 lane 2, . DEX didMaterials and Methods). In wild-type HEK293 cells, E-64d did not affect the insulin-stimulated increase in pAkt upon DEX treatment on D3 . First, DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Second, the effects of DEX on tau phosphorylation and μ-calpain activation were prevented by LiCl pre-treatment. Third, both LiCl pre-treatment and calpain inhibition by E-64d prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.Numerous potential mechanisms have been proposed to explain the pathogenic mechanisms of IR in vivo and in AD patients warrants further research. Nevertheless, our study suggests that i) the GCs-induced tau phosphorylation may not be mediated by inhibition of insulin signaling, and ii) the GCs-induced tau phosphorylation could function upstream as a mediator of IR.Previous rodent studies suggest that IR or lack of insulin signaling can result in tau hyperphosphorylation Clinical observations suggest that DM2 is among the risk factors for AD Although the inhibitory effects of DEX were evident in both cell lines on D6, calpain inhibition did not prevent the inhibitory effects in either of the cell lines . In contHowever, how the GCs-induced tau phosphorylation mediates μ-calpain activation is not known. The activation and regulation of calpain activity in vivo is not well understood, although it is a ubiquitously expressed protein with limited proteolytic activity and therefore it is assumed to be tightly regulated In conclusion, we have demonstrated that the GCs-induced tau phosphorylation and consequent μ-calpain activation may contribute to IR. Our study suggests that tau phosphorylation and μ-calpain activation may be two key events that link GCs, IR and AD.


Dynamic protein phosphorylation is an essential regulatory mechanism in various organisms. In this capacity, it is involved in a multitude of signal transduction pathways. Kinase-specific phosphorylation data lay the foundation for reconstruction of signal transduction networks. For this reason, precise annotation of phosphorylated proteins is the first step toward simulating cell signaling pathways. However, the vast majority of kinase-specific phosphorylation data remain undiscovered and existing experimental methods and computational phosphorylation site (P-site) prediction tools have various limitations with respect to addressing this problem.To address this issue, a novel protein kinase identification web server, PKIS, is here presented for the identification of the protein kinases responsible for experimentally verified P-sites at high specificity, which incorporates the composition of monomer spectrum (CMS) encoding strategy and support vector machines (SVMs). Compared to widely used P-site prediction tools including KinasePhos 2.0, Musite, and GPS2.1, PKIS largely outperformed these tools in identifying protein kinases associated with known P-sites. In addition, PKIS was used on all the P-sites in Phospho.ELM that currently lack kinase information. It successfully identified 14 potential SYK substrates with 36 known P-sites. Further literature search showed that 5 of them were indeed phosphorylated by SYK. Finally, an enrichment analysis was performed and 6 significant SYK-related signal pathways were identified.http://bioinformatics.ustc.edu.cn/pkis.In general, PKIS can identify protein kinases for experimental phosphorylation sites efficiently. It is a valuable bioinformatics tool suitable for the study of protein phosphorylation. The PKIS web server is freely available at  Reversible protein phosphorylation, which is one of the most common post-translation modifications in eukaryotes, is involved in various cellular processes including regulation of metabolism, DNA rep32P-labeling and degenerate peptide library screening, are costly, labor-intensive, and time consuming[In recent years, considerable efforts have been devoted to experimental and computational identification of phosphorylation data. Historically, phosphorylation sites were discovered mainly using low-throughput technology. Howeveronsuming,10. Withonsuming. Howeveronsuming. This lionsuming. Howeveronsuming, KinasePonsuming, Musiteonsuming and GPS2onsuming, may genTo address these limitations, this study presents a novel bioinformatics tool called the protein kinase identification server (PKIS). It is designed for the identification of protein kinases that act at known P-sites with high specificity. Human phosphorylation data was retrieved from the Phospho.ELM database and used to train the kinase identification models by incorporating the composition of monomer spectrum (CMS) with SVMs. ComprehPrevious studies have demonstrated that the side chains of amino acids surrounding P-sites influence the phosphorylation process, including contacts with kinases. In this way, the sequence surrounding a P-site plays a vital role in determining which kinase catalyzes the corresponding phosphorylation substrate. However, the residues enclosing the P-sites in the linear sequence may not be adjacent spatially, and distinguishing the residues surrounding the P-sites exactly for all the phosphorylated proteins experimentally is difficult and time-consuming. For them is equal to 8, but it soars to 0.842 when m increases to 16. The improvement in AUC begins to slow down as window size increases beyond 16, finally stopping at the maximum value of m. This indicates that all the residues implicated in the phosphorylation process have been taken into account. It is of note that there are some fluctuations in improvement as the window size increases, probably due to complex interactions between residues.In light of kinases’ specificity in protein phosphorylation, it does not make sense to use a fixed window size for all kinases. For thim is equal to 19. For this reason, this optimized window size was used to build the SVM model for MET kinase in PKIS.To identify protein kinases confidently, the specificities of the SVM models in PKIS were all required to be at least 99.0%, which meant that the expected rate of false positive results was not larger than 1.0%. Then changes in sensitivity at different window sizes were examined  in CMS encoded features, which is consistent with the disparity of amino acid compositions between positive and negative data. These results demonstrate that CMS-encoded features are useful for the determination of which kinase catalyzes the corresponding phosphorylation process.An essential part of developing a protein kinase identification system is the encoding of the side chains surrounding the P-sites. A good, high-performance encoding strategy may also provide insight into the biological mechanism of phosphorylation. First, we examined the features encoded by CMS that represent different amino acid compositions under a series of increased window sizes. Amino acid compositions were found to be largely different for the positive and negative data in most of the kinases. For example, Figure Another encoding strategy, binary encoding was also investigated. Binary encoding is widely used in bioinformatics studies of protein phosphorylation. In binary encoding, a 21-dimensional binary vector represents each amino acid and an end-of-sequence marker. Phosphorylation data were encoded based on CMS and binary strategies and the performance of these two methods was evaluated using LOOCV. The ROC curves for CK2alpha and CDC2 kinase were used as examples Figure . AccompaThe performance of PKIS was evaluated and compared to three widely used kinase-specific P-site prediction tools: KinasePhos2.0, MusiteTo evaluate performance at high specificity, a threshold for decision scores and probabilities returned by P-site prediction tools was used to ensure that specificity levels fell as closely to 99.0% as possible. As shown in Figure To assess the performance of the PKIS web server, the phosphorylation data from high-resolution maps of the human phosphorylation network were extracted and a testing dataset of experimentally identified kinase-substrate pairs was constructed. To makeP-values below 0.05. The most significant pathway is associated with natural-killer-cell-mediated cytotoxicity . Cytotoxicity mediated by natural killer cells is a very important immune response, playing both anti-viral and anti-tumor roles[Increasing knowledge of P-sites and their corresponding protein kinases is critical to reconstructing signal transduction pathways. In the present study, PKIS was used to identify P-sites phosphorylated by SYK kinase, which has been reported to mediate various cellular processes. There aor roles. In thisor roles. In thisor roles-32. Theshttp://bioinformatics.ustc.edu.cn/pkis/. Users can submit phosphorylated proteins with verified P-sites and select all or some of the 56 predictive models available for protein kinase identification. In Additional filehttp://bioinformatics.ustc.edu.cn/pkis/download.html.PKIS is freely accessible to users at the following web address: in vivo. As such, the primary sequences around the potential P-sites may be not sufficient to indicate the corresponding protein kinase. The performance of this system may be enhanced by incorporating more biological information, such as protein functional domains and subcellular localization. Currently, kinase-specific phosphorylation data for other organisms are still sparse. However, with rapidly accumulated phosphorylation data, it may be possible to develop a platform that can be used to accurately identify protein kinases in multiple organisms.Protein kinase identification is attracting significant attention due to the large number of P-sites discovered using high-throughput technologies. In the present study, a novel kinase identification web server was developed based on CMS encoding strategy and SVMs. In addition, to achieve optimal performance we generated specific negative data for SVM training in that different negative dataset construction strategies can bring about significantly different performance with respect to the classification problems. The resAll 37,145 phosphorylation instances in humans were extracted from the latest version of Phospho.ELM (9.0). After excluding redundant records, 27,404 P-sites were recognized in 5,374 proteins, including 3,151 kinase-substrate pairs. These phosphorylation sites and their kinase information were used for further analysis. For each kinase, the corresponding phosphorylation instances were used as positive data (+). Negative data (−) were comprised of phosphorylation events catalyzed by other kinases, instead of non-phosphorylation sites that were used by P-site prediction tools. To ensure reliable results, a total of 56 kinases with more than 10 positive instances were selected. See Additional filem, CMS incorporates different MS vectors under a series of window size from 3 to 2*m+1. Unlike the MS encoding strategy, this reflects the occurrence of the amino acids in certain positions and therefore provides more sequential information than amino acid composition for given window size. Additional fileIn this study, sequence information was encoded using an efficient encoding strategy called CMS. As a pac) value and the gamma (γ) value were optimized and used to enhance the strength of the classifiers. Leave-one-out cross validation , which is the most objective and rigorous method of assessing a classifier, was used to evaluate the performance of PKIS. The two performance measurements adopted in this study are defined as follows:The kinase identification system was constructed by incorporating SVMs with CMS features. LIBSVM, a public SVM library, was selected for training classification models. The radThe receiver operating characteristic (ROC) curves were also plotted and the area under the curves (AUC) was calculated as additional measurements of performance. To minimize possible false positives in the results, for each prediction, a threshold was adopted to guarantee that the specificity was no less than 99%. For each kinase, the optimal window size used to encode CMS encoding was determined using the best sensitivity obtained in LOOCV.The authors declare that they have no competing interests.MHW and LZ conceived the idea of this work. MHW supervised the whole project and participated in its design and coordination. LZ and MHW developed the algorithm, implemented programs and drafted the major of the manuscript. MW developed the PKIS web server and helped to draft the manuscript. YS, JL and AL carried out the interpretation, reviewed the study and provided biological insights. All authors read and approved the final manuscript.Multivariate analysis of variance (MANOVA) of the CMS encoded features.In total, 50 kinases pass the statistical test as dictated by the requirements of MANOVA.Click here for fileComparison of PKIS with kinase-specific P-site prediction tools using the Phospho.ELM database. Some P-site prediction tools (such as KinasePhos) do not report scores for P-sites that are predicted to be unphosphorylated. To plot ROC curves, the scores of these P-sites were set at 0, which may sometimes lead to vertical ROC curves (dashed lines). Note that, in this case they may not precisely represent real performance of protein kinase identification processes.Click here for fileCross-classification of specificity among 40 Ser/Thr kinases based on kinase identification models.Click here for fileCross-classification of specificity among 16 Tyr kinases based on kinase identification models.Click here for fileSequence logos of amino acids surrounding phosphorylation sites catalysed by four kinases. The horizontal axis represents sequential positions relative to phosphorylation sites and the vertical axis represents decreases in uncertainty. Each letter denotes one amino acid.Click here for fileCross-classification of performance of protein kinases with similar substrate motifs. Some kinase-specific P-site prediction tools (e.g. Musite) cannot distinguish different protein kinases within the same kinase group. For this reason, in this case, cross-classification is not applicable (NA).Click here for fileA screen capture of a prediction made using PKIS. Two protein sequences were used in this example. PKIS also provides the CMS logo of each kinase, which contributes to better understanding of the substrate binding preference of each protein kinase.Click here for fileStatistics of all kinases in PKIS. The PKIS provides 56 kinases with more than 10 positive instances.Click here for fileDifference between CMS and MS encoding strategies. Two different sequence encoding strategies were used. For the sake of simplicity and clarity, a sequence of 5 amino acids served as an example. Panel (A) shows the monomer spectrum (MS) encoding strategy. Panel (B) shows the composition of monomer spectrum (CMS) encoding strategy.Click here for file


Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells. During acute inﬂammation, leukocytes are recruited from the circulation to sites of infection and injury. This multistep adhesion and signaling cascade is initiated by interactions between selectins and their glycoconjugates that mediate leukocyte tethering to and rolling on the surface of endothelial cells P-selectin Like other cell adhesion receptors, selectins and their ligands also transduce signals The goal of the present work was to determine whether L- and E-selectin form oligomers or monomers on cell surfaces. We used the micropipette adhesion frequency assay to analyze two-dimensional (2D) binding between selectins and ligands Chinese hamster ovary (CHO) cells transfected to stably express human E-selectin were previously described Neutrophils were isolated from a drop of whole blood via a finger prick and RBCs were isolated from peripheral blood drawn from healthy volunteers according to protocols approved by Georgia Institute of Technology’s Institutional Review Board Human L-selectin-Ig was expressed and purified as previously described 3) method 3 solution was prepared, aged at pH 5, and diluted in 20 mM acetate-buffered saline, pH 5.5, at ratios ranging from 1∶100 to 1∶2400. RBCs were washed five times in saline and resuspended in 1% hematocrit. Antibodies were added to each 250 µl sample and mixed. An equal volume of diluted CrCl3 solution was added dropwise with constant agitation. After 5 min the reaction was stopped by addition of 0.5 ml phosphate buffered saline plus 1% IgG-free bovine serum albumin (BSA). Aliquots from each sample were examined under light microscopy for aggregation. Cells were subsequently washed and stored in EAS45. Immediately prior to the micropipette experiment, RBCs precoated with 1D6, anti-Fc or PL2 were respectively incubated with sE-selectin (0.5 µg/ml), L- or E-selectin-Ig (0.5 µg/ml), or (s)PSGL-1 (0.2 µg/ml) at room temperature for 30 min.A previously described modified chromium chloride (CrClPa) observed in 50∼100 repeated contact cycles using a single pair of cells. Three to five cell pairs were tested to obtain a mean Pa ± S.E.M. for that contact duration (t). Measurements were made in five contact durations to obtain a Pa vs. t binding curve for each receptor-ligand pair respectively expressed on the corresponding cells at given site densities measured separately by flow cytometry.A previously described micropipette adhesion frequency assay Cell samples were incubated with saturating concentrations of fluorescence-conjugated primary antibodies (10 µg/ml or according to manufacturer’s instruction) in FACS solution (RPMI with 1% BSA and 0.05% sodium azide) for 30 min followed by three washes, and analyzed immediately. If non-conjugated primary antibody was used, cells were next incubated with FITC-conjugated secondary antibody (10 µg/ml or per manufacturer’s instruction) and then washed and analyzed immediately. Cells and calibration beads from quantum FITC MESF kits  were analyzed on a BD LSR II flow cytometer . The mean fluorescent intensities (MFIs) were recorded for samples as well as for each peak of the calibration beads. A standard curve was plotted using the MFIs of the calibration beads, which was used to calculate molecules of equivalent soluble fluorophore (MESF) from the MFIs measured from the cells. Antibodies bound per cell were then calculated by dividing MESF by fluorophore per antibody (supplied by manufacturers), which were then converted to site densities by dividing by the cell surface area The capacity of capturing E- or L-selectin-Ig by goat anti-human IgG antibody precoated on RBCs was measured using a FITC-conjugated human IgG. At saturating concentration, each binding site for Fc should capture one dimeric E- or L-selectin-Ig. Direct measurement using anti-E-selectin (ES1) and anti-L-selectin (DREG56) mAbs obtained similar results. The site density of sE-selectin was controlled by the site density of its capture antibody (1D6) precoated on RBCs, which was measured using FITC-labeled goat anti-mouse IgG mAb A85-1. The site density of sPSGL-1 was measured using PE-labeled anti-PSGL-1 mAb PL1. Since the same batch of RBCs pre-coated with the same density of PL2 were incubated with saturating concentrations of PSGL-1 or sPSGL-1, only one leg of PSGL-1 would be captured by PL2. Thus, the site density for dimeric PSGL-1 was inferred to be the same as that of monomeric sPSGL-1, whereas the density of selectin binding-sites of PSGL-1 should be twice as that of sPSGL-1. It is possible that a fraction of PL2 bound dimerically with the dimeric PSGL-1 even though a saturating concentration was used. Should this be the case, using our indirect inference would overestimate the selectin binding sites on PSGL-1-coated RBCs. This would increase, rather than decrease, the confidence of our interpretation regarding the formation of dimeric bonds from the observation of higher selectin binding to PSGL-1-coated RBCs than to sPSGL-1-coated RBCs. The site densities of E-selectin on CHO cells and HAECs and of L-selectin on neutrophils were measured using anti-E-selectin mAb ES1 and anti-L-selectin mAb DREG5.in situ kinetic analysis, we determined the ability for endothelial cell E-selectin and neutrophil L-selectin to support dimeric interactions by comparing their binding properties with PSGL-1 and sPSGL-1.To discriminate the multimericities of E- and L-selectin on their respective cell surfaces, we used the micropipette adhesion frequency assay, which measures cross-junctional ligand interaction with a cell surface receptor in its native membrane environment Pa vs. contact duration t curves for RBCs coated with matched densities of either sPSGL-1 or PSGL-1 and RBCs coated with sE-selectin at two site densities PSGL-1 binding to sE-selectin were indistinguishable regardless of the sE-selectin site densities used , A and BThe results of the preceding section indicate that 1) dimeric interactions required both receptor and ligand to be dimeric; 2) dimeric interactions formed more frequently than monomeric interactions; and 3) E-selectin had the same affinity with each leg of dimeric PSGL-1 as with monomeric sPSGL-1. We next compared the 2D binding of sPSGL-1 and PSGL-1 to E-selectin on the surface of two cell types: HAECs stimulated by IL-1β to induce native E-selectin expression at two levels  and CHO Similar to We next employed the same strategy to analyze L-selectin. In contact durations from 0.125–2 s, RBCs bearing either of two site densities of dimeric L-selectin-Ig adhered to RBCs bearing dimeric PSGL-1 more frequently than to RBCs bearing monomeric sPSGL-1 . This isWe lastly compared the 2D binding of sPSGL-1 and PSGL-1 at two matched site densities to L-selectin constitutively expressed on human neutrophils. Nonspecific adhesions were controlled by using RBCs precoated with an anti-PSGL-1 capturing mAb PL2 but not further incubated with either form of PSGL-1, which abolished binding . In contUsing adhesion frequency measurements, we have demonstrated that E-selectin on endothelial cells or transfected CHO cells supports dimeric interactions with dimeric PSGL-1, whereas L-selectin on neutrophils supports only monomeric interactions with dimeric PSGL-1.Our observed monomeric interactions of L-selectin with PSGL-1 are consistent with molecular stiffness measurements that L-selectin from human tonsils reconstituted into lipid bilayers forms monomeric bonds with PSGL-1 cis interactions of E-selectin in the membrane enable dimeric bonds with PSGL-1. How an E-selectin dimer is stabilized on a cell is an interesting question for future studies.Our observed dimeric inteactions of E-selectin with PSGL-1 differ from previous observations that E-selectin from lysates of CHO cell transfectants reconstituted into supported lipid bilayers forms monomeric bonds with PSGL-1, although P-selectin from lysates of human platelets reconstituted in the same lipid bilayers forms dimeric bonds with PSGL-1 123IIbMany adhesion molecules are constitutively expressed on the cell surface as homodimers or oligomers. In addition to P-selectin and PSGL-1, the βIIbβ3 integrin Receptor oligomerization is usually demonstrated by biochemical methods, e.g., chemical crosslinking, as in the case of αOne limitation is the resolution of the our methods. The biomechanical assays based on pico-force techniques usually have sufficiently high resolutions for mechanical variables such as bond lifetime, molecular stiffness, cell rolling velocity, bond rupture force, and adhesion frequency. However, they must be combined with assays to measure site densities of the interacting molecules. Biochemical assays sometimes suffer from inter-experimental variations, as measurements depend on the activities of the reagents that can vary in each preparation. This may explain the apparent discrepancies between To support dimeric interactions with dimeric PSGL-1, endothelial cell E-selectin must form oligomers of at least two proteins. If E-selectin forms oligomers larger than dimers on the endothelial cell surface, our data cannot determine the actual size of the oligomers. This limitation can be overcome by using oligomeric ligands of larger sizes to dissect the size of E-selectin oligomers combined with mathematical modeling to relate the binding curve to 2D kinetics rates. With such extensions, the present method has the potential to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells and extract intrinsic kinetic coefficients for such multimeric interactions.In addition to molecular self-association, selectins cluster in specific membrane domains through interactions with other cellular elements. L-selectin clusters in the tips of leukocyte microvilli, which enhances tethering under flow


Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞ − [P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex  against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.Inactivation of purified  A folded protein does not exist in a single conformation, rather as a set of related conformations whose interconversion involves the making and breaking of the weak  interactions such as hydrogen bonds, van der Waals, salt bridges, and hydrophobic interactions that stabilize the folded structure of the protein. The range and speed of interconversions between the conformations will depend on the magnitudes of the relevant energy barriers. In essence therefore, the observed three-dimensional structure of a protein should be viewed as a weighted average of all the conformations accessible on the time scale in question  in speciUnfolded peptides, polypeptides, and chemically unfolded proteins are flexible –17. FlexLigand binding not only increases the rate at which denatured enzymes regain their activity during renaturation in their presence, but also maintain the native conformation of proteins during denaturation in their presence. This suggests that ligands act as a folding nucleus about which the remaining constructed regions are easily induced to assume a more biologically active conformation . Recentlβ-galactosidase in the presence of urea was effected via different inhibition patterns. While folding is modulated by the solvent environment [S] on A, noncompetitive inhibition is involved when A is independent of [S], while a straight line will be obtained either in the plot of 1/A against [S] for competitive inhibition, or 1/A against 1/[S] for uncompetitive inhibition. Alternatively, from the effect of [S] on [P]∞, a competitive inhibition is predicted when a plot of [P]∞ against [S] gives a straight line passing through the origin. For noncompetitive inhibition, the plot of 1/[P]∞ against 1/[S] will be a straight line whereas for uncompetitive inhibition [P]∞ will be independent of [S]. In this work, we study the binding of galactose to β-galactosidase in the presence of the denaturant, GdnHCl, and end with a summary of the import of our findings to the current knowledge of protein folding. Besides, we also contrast the relevance of our present findings from our previous work t  were plotted for 0, 3 M-GdnHCl  formed at any time interval t, was directly related to the substrate concentration, [S] ([pNPG]). With increase in reaction time t, [P]t approached a constant value [P]∞, at each [pNPG]. However, in the presence of the 3 M GdnHCl, the [P]∞ for each [S]o decreased with respect to that in the absence of the [GdnHCl]  versus time t  versus time plots. Plots of A versus [S]o in the absence of galactose was zero order  had the highest A value when compared to those for the [galactose]. However, with respect to the [galactose], the value of A rose slightly from 5 mM through 10 mM to 20 mM . At this concentration of galactose as determined from the plot of k+o, k+o′ versus [galactose], the dominant inhibition mechanism is the binding of the galactose to the enzyme via a competitive mode of inhibition. Characteristic of competitive inhibition was an increase in Km, while the decrease in Vmax shows that the inhibition mechanism was not wholly competitive. A reviewer of this paper brought to our notice a possibility that our results could also be due to nonproductive binding variant of competitive inhibition.In the presence of GdnHCl, the [r ([P]∞/[E]o) and the highest Km and Vmax values. Also at 10 mM galactose, there was an enhanced non-competitive inhibition mechanism when compared to the 5 mM galactose as seen from the intercepts on the y-axis of the plots of [P]∞ versus [S]o. One consequence of a high Km is decrease in substrate specificity and decreased binding affinity, as a result of unstructuredness and increased flexibility of the enzyme active site induced by the GdnHCl. When Km > [S]o, the enzyme binds the substrate weakly and, therefore, the [ES] is present only at low concentrations. It has been suggested that, for the low Km binding processes with the tightly bound substrate, the ground state of the reaction is the ES complex, and the activation energy of the reaction is higher than for the high Km binding processes involving a weakly bound substrate in which the ground state of the reaction is the free reactants o would prevail over the rate of product formation when compared with 10 mM galactose which was less competitive and more non-competitive. The 20 mM galactose exhibited, essentially, a non-competitive inhibition mechanism. As could be deduced from the plot of Km or Vmax versus [galactose], the 10 mM galactose signified the transition between a predominantly competitive (below 10 mM) and a predominantly non-competitive (above 10 mM) inhibition pattern. Although the 10 mM galactose had a higher Vmax compared to the 20 mM galactose, the 20 mM still had a higher partition ratio, r, compared to the 10 mM galactose. This is not surprising since the galactose appears to enhance the effect of the GdnHCl on the free enzyme, although this effect appears inversely proportional to the [galactose]  in the absence of galactose, however, it could not induce native structural reorganization that favored the native state formation. Furthermore, the plot of A versus [S] for the GdnHCl was a zero order plot showing that on a global scale the substrate could not protect the enzyme; rather, it did so only at a local level (within the active site region).Compared to the 5 mM galactose, the 10 mM galactose had a higher partition ratio, eactants . Thus, aeactants . As highalactose . In factS] leads to catalysis via the modulation of the binding funnel towards reduced energy states with lower entropy, but with no apparent effect on the folding funnel. In other words, within the conformational equilibrium induced by the 3 M GdnHCl, the substrate binds and displaces the equilibrium towards a conformer or conformers with appropriate catalytic activity without the need to initiate global refolding. In the case of galactose, in an apparent paradox, even though the galactose (up to 10 mM) potentiated the inhibitory/inactivating effect of the GdnHCl on catalysis probably by synergism, it simultaneously modulated folding to a reduced ensemble of states as was seen by the decreased A with respect to the absence of galactose and 3 M GdnHCl. Above 10 mM, the full folding potential of galactose was realized, despite its inhibitory ability. Thus, a bit of catalytic proficiency was sacrificed for folding. In relation to the energy landscape, binding of the , it is clear that the various conformers have a high kinetic barrier between them since their KmS vary considerably. The ligand as a function of its concentrations binds to conformers most precise to a given concentration  and “pulls” the equilibrium via the dynamism of the energy landscape to favor the given conformer. We also suggest that since the k+o and the k+o′ decreased as the mode of inhibition became increasingly non-competitive  while the A increased, then there must be some sort of induced fit mechanism which propagates from the site of binding , the dominant binding mode of inhibition would have to involve competitive inhibition. In the case of GdnHCl, in addition to possessing amino (as does urea) and imido groups, the denaturant possesses HCl which is strongly electrostatic/polar . Tsai and Nussinov ∞/[S]o ratio any given time interval, t, in comparison to the presence of the galactose, while still possessing the highest A. Even though our A values of 0.0161 s−1–0.0323 s−1 compare favourably well with a value of 0.016 s−1 for papain, unlike in the work of Tian and Tsou [P]∞ versus [S]o for the 3 M GdnHCl shows that the GdnHCl was interacting via a non-competitive binding mode with the enzyme that is away from the enzyme active site. However, from the nature of the slope of the [P]∞ versus [S]o plots for the GdnHCl alone, in comparison, to those in the presence of galactose, it becomes clear that site specific binding might not have been the only possible mode of binding for the GdnHCl. It is suggested that the solvent-exchange mode of interaction, in which the interactions of both the solvent (buffer) and the cosolvent (GdnHCl) with the protein involve the interchange between the components at a particular interaction “site” on the protein  in a local minimum which topologically favours the formation of a native state as a result of coupled binding and catalysis, as mediated by the ligand nature and concentration.It is seen from β-galactosidase via the ligand concentration when unstructuredness is induced by a denaturant such as urea or GdnHCl. Below is a scheme suggesting the interconvertibility of the binding mode of S are the enzyme forms that apparently have the least ΔG at a given [galactose] in a local minimum which topologically favours the formation of a native state as a result of coupled binding and catalysis, as mediated by the ligand nature and concentration.From opposite binding and inhibition modes at same [galactose]. They do this basically by their interactions with the protein and varying [galactose], which results in different forces. It is these forces that determine the outcome of the interactions reflected as different inhibition mechanisms . The cosms  and can function in its current state, then its attained Gibb's free energy for that state becomes its minimum even if it is in a metastable state. Thus, in vivo, many conformers of an enzyme may exist within a conformational/metabolic enclave, so that subtle environmental factors become the important parameters in a selection process making “the native state” a relative term and idiosyncratic. Our results suggests that a so-called β-galactosidase probably exhibited the morpheein concept—the ability of a homooligomeric protein to exist as an ensemble of physiologically significant and functionally different alternate quaternary assemblies—coming apart and changing shape so as to convert between forms [β-galactosidase exhibited moonlighting protein properties, peculiar to intrinsically unstructured proteins (IUPs) (but certainly not restricted to IUPs) where such proteins are able to fulfill more than one, apparently unrelated function using different sites [β-galactosidase to moonlight and at the same time morph by breaking down into varying secondary structures, at different times, so as to convert into entirely new forms with different functionalities? Could it also be possible that β-galactosidase performs its metabolic regulatory activities [C-value paradox [Jaffe's group  suggesteen forms . Hystereen forms . Howeveren forms , β-galacnt sites . This rativities  via the  paradox  to accouSmall molecules as effectors  due to  We end our write-up with a summary of the relevance of our findings to the current knowledge of protein folding just as we contrast the relevance of our present findings from our previous work .Even thoIn conclusion, the possible implications of our findings in vivo in biological systems might include (1) that enzyme catalytic sites (as the binding funnel) and hydrophobic cores (as the folding funnel) could be modulated by the ligand to give rise to different stable conformations of the enzyme. (2) The binding mode, which drives folding, is determined by different forces arising from interaction sites. (3) Biological processes are carried out through binding, which respond to changes in the solvent environment. In different enzymes, the binding behaviour varies as a result of differences in structure/function and the environment.


Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on  Toxoplasma gondii is an obligate intracellular apicomplexan parasite that is an important pathogen of humans and animals. T. gondii causes encephalitis in immunocompromised patients, and progressive encephalitis in children infected in utero T. gondii consists of two phases: the sexual, which takes place only in felines, and the asexual, which takes place in all mammalian and avian hosts Toxoplasma infections T. gondii drugs are urgently needed. Studies on the basic biology of this organism are thus necessary for discovery of novel targets, and may also serve as a model system for the study of other apicomplexan parasites.Eukaryotic signaling pathways regulate a spectrum of intracellular activities; for example, the cAMP-dependent pathway is known to influence gene expression, apoptosis, tissue differentiation, and cellular proliferation Plasmodium falciparum, cAMP has been implicated in the differentiation of gametocytes Leishmania majorGiardia lambliaSchistosoma mansoniT. gondii, studies using cAMP analogs and cyclic nucleotide phosphodiesterase (PDE) inhibitors, which inhibit inactivation of cAMPs, implicated cAMP signaling in the asexual stage of the parasite T. gondii, whether these effects are extracellular , intracellular , or due to stimulation of both host and parasite signaling pathways remains to be determined. Moreover, although H89 is considered to be a specific inhibitor of PKA, several recent reports have suggested that H89 inhibits other kinases such as mitogen- and stress-activated protein kinase 1, p70 ribosomal protein S6 kinase or Rho-dependent protein kinase II Both cAMP and PKA have been shown to be essential signaling components in the life cycles of many eukaryotic pathogens. In in vitro kinase assay using recombinant T. gondii PKA catalytic subunit (TgPKA-C), as well as tachyzoite growth assay. The effect of PKI was weaker in TgPKA-C compared to mammalian PKA-C in the in vitro kinase assay, and PKI did not inhibit tachyzoite growth. These data support the hypothesis that the inhibitory effect of H89 on tachyzoite growth is due to TgPKA-C inhibition, since the possible inhibition of host cell PKA-C activity by PKI did not result in the inhibition of tachyzoite growth. Moreover, we generated a parasite line that expressed TgPKA-R stably, in which inhibiting the activity of parasite PKA without any effect on host cell PKA might be possible. These parasites also showed decreased growth. According to these data, TgPKA-C does indeed play an important role in the asexual stage of the T. gondii life cycle.In this study, we compared the inhibitory effects of ATP competitive chemical inhibitor H89 and mammalian natural peptide inhibitor PKI in an P. falciparum and Homo sapiens. The putative TgPKA-C (343 amino acid residues) showed 54% identity with H. sapiens PKA-Cα  and 57% identity with P. falciparum PKA-C . A multiple alignment using the ClustalW program showed that the 11 major subdomains of protein kinases (I–XI) The amino acid sequence alignment of the putative TgPKA-C , which we identified for the first time, is shown in P. falciparum and H. sapiens, is shown in H. sapiens PKA regulatory subunit Iα , 35% identity with HsPKA-RIIα (GenBank Accession Number: NP_004148), 36% identity with HsPKA-RIβ (GenBank Accession Number: NP_001158230), and 32% identity with HsPKA-RIIβ (GenBank Accession Number: NP_002727), as well as 52% identity with P. falciparum PKA-R . TgPKA-R possessed the two most conserved regions of PKA-R: the phosphate-binding cassettes (PBCs) of cAMP-binding domains A and B, specific to proteins of the PKA-R family r of 53,000 or 69,000, respectively, as detected by silver staining. These proteins reacted with antiserum containing α-GST antibody. These data indicate that we successfully purified the desired GST fusion proteins.To characterize the function of the protein, we expressed and purified TgPKA-C as a GST fusion protein using a wheat germ cell-free protein synthesis system. Purified protein was then electrophoretically separated in a denaturing gel and either silver-stained or immunoblotted with rabbit antiserum containing α-GST antibody . The purr of 69 000 was labeled  in accordance with the manufacturer's instructions. The labeled TgPKA-C band was eliminated by phosphatase treatment, indicating that TgPKA-C was labeled with [γ-32P]ATP via phosphorylation  was included as a positive control. Compared to BtPKA-C, inhibition by PKI was much weaker in TgPKA-C . However, in the experiment in which P. falciparum lysate containing cAMPs was used instead of recombinant TgPKA-C, phosphorylation of PfPKA-C markedly decreased The mechanism by which TgPKA-R, overexpressed in transgenic parasites, inhibited the activity of natural PKA-C is unclear. Two possibilities exist: TgPKA-R may act as a pseudosubstrate of TgPKA-C by inhibiting its binding to a substrate, or it may act as a cAMP sink, thereby reducing available cAMP so that PKA-C cannot be dissociated from PKA-R. When we used GST-TgPKA-C and MBP-TgPKA-R under cAMP-free conditions in an T. gondii. Several proteins have recently been reported to interact with PKA-C in apicomplexan parasites. In P. falciparum, the merozoite transmembrane protein AMA1, which is involved in the invasion of erythrocytes, has been reported to be phosphorylated by PKA T. gondii possesses a homolog of this protein (TgAMA1), TgPKA may also interact with it, thereby regulating invasion. In Trypanosoma cruzi, yeast two-hybrid experiments revealed that several proteins with known functions interact with PKA T. cruzi growth, adaptation, and differentiation. Since some of these proteins, for example, hexokinase and aquapoline, also exist in T. gondii, investigating their interaction(s) with TgPKA-C might be interesting.Further understanding of the biology of TgPKA may best be gained by investigation of the pathways and proteins with which TgPKA-C interacts. Since PKA is known to be involved in various activities of mammalian cells, many types of substrate may also be present in T. gondii RH strain and RH/ht− strain (kindly provided by Dr. X. Xuan) were used in this study. Parasites were maintained in monolayers of Vero cells cultured in Dulbecco's modified Eagle's medium (DMEM) that contained 7.5% fetal calf serum (FCS), 2 mM l-glutamine, 20 mM HEPES (pH 7.5), streptomycin, and penicillin. Host Vero cells were passaged in the same medium.Tachyzoites of T. gondii RH strain mRNA was isolated from infected Vero cells using the TRIZOL reagent  according to the manufacturer's instructions. The entire TgPKA-C open reading frame (ORF) was amplified by RT-PCR using parasite mRNA as the template and the following primers: forward, 5′-CCGGCTCGAGGATCTTTTTCGCCGTCTAAT-3′ and reverse, 5′-CGGGATCCTTACCACGACGTGAAAGGGT-3′. The PCR product was digested with XhoI/BamHI and cloned into the XhoI and BamHI sites of pEU  to express a glutathione S-transferase (GST) fusion protein. The resultant plasmid was designated pEU-GST-TgPKA-C. The entire TgPKA-R ORF was amplified from parasite mRNA by RT-PCR using the primers 5′-CGAATTCGGTAACGTTTGTGAATGCCAAAAG-3′ and 5′-GGGCTGCAGTTATTTGTCGAGGTATTTGGTGTCG-3′. Amplified fragments were digested with EcoRI/PstI and cloned into the EcoRI and PstI sites of pMal-c  in-frame with maltose-binding protein (MBP), to generate pMal-TgPKA-R. For stable expression, pMFH-TgPKA-R was generated as follows. The TgPKA-R ORF attached to the EcoRI/EcoRV restriction site was amplified from pMal-TgPKA-R using the primers 5′-CGAATTCGGGTAACGTTTGTGAATGCC-3′ and 5′-GGATATCTTTGTCGAGGTATTTGGTGTCG-3′. Amplified fragments were digested with EcoRI/EcoRV and cloned into the EcoRI and EcoRV sites of pMini.3×Flag.ht µg of pEU-GST-GFP or pEU-GST-TgPKA-C was mixed with 18 µL transcription mixture  and incubated at 37°C for 6 h. Each mRNA generated was then mixed with 10.8 µL WEPRO1240G (CellFree Sciences) and 40 ng/µL creatine kinase , then transferred to the bottom of the SUB-AMIX (CellFree Sciences) to form a bilayer and incubated at 16°C for 20 h.Protein expression using the Wheat germ cell-free protein synthesis system (CellFree Sciences) was performed as described previously µL of a 50% slurry of glutathione sepharose beads  for 16 h. The beads were then washed three times with phosphate-buffered saline (PBS)(–). Purified protein captured on the beads was separated by 10% SDS-PAGE after boiling and either silver-stained or immunoblotted  at 37°C for 2 h were pelleted and solubilized in 1% Tween 20/PBS(–) using one freeze–thaw cycle and three 10 s bursts of ultrasonication. After centrifugation , the supernatant was mixed with amylose resin beads (New England BioLabs) at 4°C for 2 h, and the beads washed five times with 1% Tween 20/PBS(–).Wheat germ extracts were mixed with 15 oblotted  with α-GµL kinase buffer  containing 5 µCi [γ-32P] ATP. Samples were then washed with TNE buffer . Reactions were performed at 30°C for 30 min in a total volume of 50 Following the kinase assays, labeled GST fusion proteins were boiled to inactivate the kinase and incubated with λ-protein phosphatase (New England Biolabs) in accordance with the manufacturer's instructions.T. gondii RH/ht− was performed as described previously µg of plasmid DNA was electroporated into 5.0×107 parasites using a GenePulser  at 2000 V, 25 µF, and 50 ohm. At 24 h after transfection, parasites were selected by exposure to 50 µg/mL mycophenolic acid and xanthine for 48 h, followed by a subsequent plaque purification. The recombinant clones stably expressing GFP or TgPKA-R-3xFlag were designated RH/GFP or RH/TgPKA-R.Transfection of the expression plasmid to 14–22  was 2 µM, and the volume of DMSO added was the same as that of H89. After incubation for 12, 24, and 36 h, the coverglass slides of RH/TgPKA-R were fixed with 4% paraformaldehyde in PBS(–) for 10 min, permeabilized by 0.1% Triton X-100 in PBS(–) for 10 min, and blocked in 3% bovine serum albumin and 5% skim milk in PBS(–). For the immunofluorescence assay (IFA), the slides were incubated with mouse α-SAG1 (tachyzoite-specific surface antigen) polyclonal antibody  at a 1∶1000 dilution for 1 h at room temperature. After reaction with the primary antibody, slides were washed three times with T-PBS [0.1% Tween 20 in PBS(–)], and primary antibody was detected with Alexa 546-conjugated α-mouse antibody (Invitrogen) at a 1∶2000 dilution for 1 h at room temperature, followed by five washes with T-PBS. The coverglass slides of RH/GFP were fixed with 4% paraformaldehyde in PBS(–) for 10 min and then washed three times with PBS(–). Each coverglass slide was dried at room temperature, mounted under a coverglass using Fluoromount-G , and observed under a fluorescence microscope . Microscopic fields were chosen randomly to count parasitophorous vacuoles up to three hundred. Vacuoles were sorted by the numbers of tachyzoites inside, and average numbers of tachyzoites per vacuole were calculated. Parasite growth was determined by the number of parasites per vacuole. The representative data of several experiments are shown in RH/TgPKA-R or RH/GFP was inoculated onto confluent Vero cell monolayers growing in four-chambered coverglass slides  at a multiplicity of infection (MOI) of 10 and allowed to invade host cells for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without the PKA-specific inhibitor. The final concentration of inhibitors H89 (Promega) and PKI