Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function.Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening.Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease.Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease. Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant  The molIn vitro studies performed by Stull et al. (2+ binding to troponin C (TnC) is not the only process that regulates striated muscle contraction. Furthermore, in vitro and structural studies have implicated the negative charge associated with phosphorylation of the RLC to structurally repel myosin heads away from the thick filament toward actin , have an influential role to play in muscle contraction mediated by phosphorylation –13. In vl et al.  have shord actin –16. Therrd actin  and/or hrd actin .Furthermore, pathological mutations to the RLC in humans are known to present as familial hypertrophic cardiomyopathies. Many of these mutations occur in and around the phosphorylatable region of the RLC and can affect the ability of the RLC to be phosphorylated, as seen in the E22K mutation among others , 19, 20.Studies have been performed to elucidate RLC phosphorylation mechanisms; genetic mutant murine models of disease have been used, either replicating mutations found in human patients or creating mutant RLCs that are unphosphorylatable to assess calcium sensitivity changes , 22–26. TM SDS-PAGE method was utilized to observe the changing RLC phosphorylation profile during heart failure progression in human patients in New York Heart Association (NYHA)-classified HF progression and in a rat model of chronic MI, which manifests as early cardiac hypertrophy and eventual heart failure.In this paper, a Phos-tagIn addition, we studied and evaluated the mechanical effect of RLC phosphorylation on permeabilized cardiac tissue. We used force-velocity (FV) and power-velocity (PV) relationships to assess the effect a physiological range of RLC phosphorylations had on the contractile characteristics of permeabilized cardiac trabeculae. This was performed during muscle shortening over a set of velocities in which the heart generates power and performs work in the physiological range.All animal surgical procedures and perioperative management were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health under assurance number A5634-01. Adult male Sprague-Dawley rats (250–300 g) underwent proximal left anterior descending coronary ligation to induce chronic myocardial infarction as described previously . Followi10 mg of rat myocardium was removed from the left ventricular endocardial region close to the apex, frozen in liquid nitrogen, and stored at −80 °C. On the day of the experiment, samples were mechanically homogenized using a pestle and mortar cooled with dry ice. Samples were then added to SDS-PAGE sample buffer. Human tissue samples were also removed from the endocardial layer of the left ventricle and prepared the same way. Porcine hearts were obtained from the Royal Veterinary College. These were rinsed in ice-cold saline and prepared for gel analysis in the same manner as rat and human myofibrils.TM incorporating 15% SDS-PAGE and PVDF membrane blotting with chemiluminescent antibody detection were used to ascertain quantitative RLC phosphorylation levels. Gels were run for 120 min and blotted for 60 min. Membranes were probed with mouse anti-RLC antibodies . Goat anti-mouse horseradish peroxidase (HRP)-linked IgG was used with a Bio-Rad electrochemiluminescence kit for band visualization in a Bio-Rad GelDoc imager. ImageJ was used to threshold and measure individual band densities.Samples were heated at 95 °C for 2 min to aid protein denaturation. Gels were subsequently loaded with 10-μl individual samples. Phos-tagg) were lysed by BugBuster MasterMix  containing EDTA-free protease inhibitor mixture , and the inclusion bodies were purified according to the manufacturer's instructions. The purified inclusion bodies were resolubilized in IMAC binding buffer  containing 6 m urea for 4 h at 4 °C under vigorous stirring. After removing insoluble components by centrifugation at 15,000 × g for 30 min, the supernatant was applied to a 5-ml HisTrap FF column  with a flow rate of 1–2 ml/min. The column was washed with 20 column volumes of IMAC binding buffer containing 6 m urea, and the protein was eluted with a step gradient of imidazole  in IMAC binding buffer containing 6 m urea. Fractions containing purified protein were pooled, and urea were removed by dialyzing three times against dialysis buffer . To remove the N-terminal histidine tag, tobacco etch virus protease was added in a 1:100 stoichiometry, and the protein was digested overnight at 4 °C. The sodium chloride concentration was adjusted to 500 mm, and tobacco etch virus proteases and remaining impurities were removed by passing through 1-ml HisTrapFF  columns. The column flow-through containing the purified rRLC was collected, and the protein was concentrated to 2 mg/ml . Protein was stored at −80 °C until further use.rRLC was expressed from pET6a vector fused to an N-terminal histidine tag and tobacco etch virus protease site. After expression, cells harvested by centrifugation  and zipper-interacting protein kinase . Fresh kinase (1 μg/ml of reaction mixture) was added to the reaction mixture every 30 min. Kinases and phosphatases were removed by molecular weight cut-off column filtration. Phosphorylated rRLC was mixed 50:50 with unphosphorylated rRLC to produce control-mimicking in vivo RLC phosphorylation. Dephosphorylated rRLC was produced by incubation with shrimp alkaline phosphatase (Fermentas) (20 units/100 μl of protein) in vitro for 60 min in kinase buffer without ATP.rRLC phosphorylation species from n kinase   containing recombinant RLC was exchanged to a labeling buffer (0.1 m NaCl and 0.1 m NaH2PO4 at pH 7.2) using PD-10 columns . Protein was subsequently incubated on ice for 2 h with N-hydroxysuccinimide-rhodamine ester . Excess dye was removed using ZebaTM desalting spin columns (Thermo Scientific). Absorbance at 555 nm for the protein dye conjugate was used to indicate the labeling ratio of dye to protein. The buffer was changed to 5 mm ATP, 5 mm EGTA, 5 mm EDTA, 10 mm imidazole, 150 mm potassium proprionate, 10 mm KH2PO4, 5 mm DTT, and 0.5 mm trifluoperazine at pH 6.5 (exchange solution) for protein exchange as previously described . The stage movements and trabecular lengths were controlled using LabView software that also recorded digitized motor and force transducer outputs during experimentation. Upon activation and after peak isometric force was attained, “slack test” measurements on each preparation were carried out. Trabeculae were slackened by rapidly reducing length to between 8 and 12% of the initial muscle length. This was performed three times with each fiber. The time of force deviation from “slack force” was determined by fitting through the initial 100 ms of the tension recovery trace. The time taken for force to rise above the zero-force level was measured in each case by fitting the initial linear phase of tension development post-slack . Vmax was calculated from the slope of the line joining the data points resulting from a plot of step length amplitude versus slack time (C and D). The rapid release component effectively slackens the parallel elastic component of the preparation, giving rise to a nearly steady force level during ramp shortening.Trabeculae were mounted in relaxing solution between hooks attached at one end to a force transducer  and at the other to a motor . ShellacFig. 1ack time . The ordack time  with ram2. Vmax is reported in muscle lengths/s. For each trabeculum, the relationship between force during shortening and shortening velocity was fit to a hyperbola previously described by Hill  was observed in tissue from the inner left ventricular wall of the rat, in comparison with the phosphorylation level in non-failing human myocardium (0.41 ± 0.05 mol of Pi/mol of RLC (n = 5)). These values are very similar to that of the pig left ventricular wall at 0.42 ± 0.02 mol of Pi/mol of RLC (n = 2). Left ventricular RLC phosphorylation during the hypertrophic remodeling phase post-MI was 0.72 ± 0.03 mol of Pi/mol of RLC (n = 5) versus healthy rat at 0.53 ± 0.06 mol Pi/mol of RLC (n = 12), and this difference was assessed as the model progressed to chronically dilated and failing phenotype (0.76 ± 0.07 mol of Pi/mol of RLC (n = 4), p < 0.05), showing a significant difference in late stage disease.Phos-tagi/mol of RLC (n = 4) to NYHA III and IV patient tissues, which show a significant increase, 0.6 ± 0.03 mol of Pi/mol of RLC (n = 4) (p < 0.05) in comparison  were evaluated. A similar trend of increased RLC phosphorylation during disease was found when we studied human heart failure tissue during progressive clinically designated disease severity. Those classified NYHA I and II show a similar trend, increasing during disease progression from a control phosphorylation state of 0.47 ± 0.05 mol of Pmparison .TM gels indicated that a total of ∼1 mol of Pi/mol of RLC is incorporated into the RLC by this treatment. Another fraction of the expressed RLC was incubated with the nonspecific phosphatase shrimp alkaline phosphatase to ensure that all RLC species were dephosphorylated.In order to study the effect of RLC phosphorylation under more controlled conditions, cardiac RLC was expressed in bacteria. The phosphorylation levels of the RLC were increased by incubation with cardiac myosin light chain kinase and zipper-interacting protein kinase. The Phos-tagin vitro phosphorylated and dephosphorylated light chains to achieve a total phosphorylation level of 0.5 mol of Pi/mol. A “reduced” phosphorylation level was achieved by exchange of the dephosphorylated RLC alone, and an “increased” RLC phosphorylation level was achieved by using the phosphorylated RLC fraction for the exchange. These differently phosphorylated rRLC species were compared with endogenous trabecular RLC “native” phosphorylation . The A-band region was measured as 1.69 ± 0.05 μm at a sarcomere length of 2.25 ± 0.02, consistent with literature values for A-band length (n = 3)  by comparing pre- and post-washout fluorescence profiles. This allowed the estimation of RLC phosphorylation state postexchange for trabeculae treated with enriched or dephosphorylated recombinant RLC species .Fluorescently labeled RLC was exchanged into permeabilized trabecular muscle preparations to visualize the spatial incorporation of these recombinant RLC species into the sarcomeric network. Fig. 4 (n = 3) , 38. ExcVmax values (derived from slack tests) and force measurements at different release velocities were combined to determine the FV relations (see A and B). These two groups did not show significant differences in most parameters (a/Po) of the FV relations was affected by control exchange. Control exchange showed a 2-fold increase in the a/Po parameter (p < 0.05).Postexchange ions see  for eachions see , A and Brameters . HoweverVmax of trabeculae (p < 0.005). Average Vmax was 66% higher in the phosphorylation-enhanced trabeculae compared with the reduced phosphorylation state (p = 0.02). The multiple-comparison statistical models did not always yield significant differences between the control group, which consisted of a 50:50 dephosphorylated/phosphorylated exchange mixture, and the reduced and enhanced phosphorylation groups (p < 0.04). The velocity at which peak power occurred (VPP) showed a similar pattern; VPP in the enhanced phosphorylation group was >2-fold faster than in the reduced phosphorylation state (p = 0.012) and showed a marginal difference from the “native” control (p = 0.04). The mean force, power, and Vmax for the 50:50 control exchange series were consistently intermediate to the reduced and enhanced phosphorylation groups  in vitro  and exchin vitro . Importa reduced , 54, 55.We show that our exchange protocol used to replace native cRLC with rRLC did not affect the FV and PV relations of the trabecular preparations , demonstVPP), Vmax, and power  the inability of the RLC to be phosphorylated is a driver for cardiac pathology; 2) increases in RLC phosphorylation may signify a compensatory adaptation to pathology in certain instances.Our results complement others that suggest that cardiac disease progression may be driven by an inability to phosphorylate RLC appropriately. Here we extend the role of RLC phosphorylation in pathophysiology to acquired heart failure post-MI, with confirmation in myocardial samples from patients with advanced heart failure and an animal model of chronic MI in rats. Thus, increased RLC phosphorylation may act as a mechanism to compensate for the loss of myofilament mechanical power generation during progression of the failing myocardium.


In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr308 by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at AktThr308 in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr308, which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr308 by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.PDK1 phosphorylates multiple substrates including Akt by PIP Although GRP78  is primarily known as a resident endoplasmic reticulum chaperone, it appears on the cell surface of many types of malignant cells 2M* to tumor cell-surface GRP78 causes its autophosphorylation activating downstream pro-proliferative and anti-apoptotic signaling cascades including RAS/MAPK and PI 3-kinase/PDK1/Akt/mTORC1/mTORC2 Tetrameric human α3 is the major signaling event triggered by cell binding of growth factors and hormones. Our previous studies suggest that α2M* functions like a growth factor triggering PI 3-kinase activation and production of PIP33 in cells are mediated by AGC kinase family members that control cell proliferation, survival, and metabolic responses 308 for Akt1) and the hydrophobic motif (Ser473 for Akt1) 308 is indispensable for kinase activity whereas phosphorylation of Ser473 enhances kinase activity by fivefold 3 binding to its PH domain promotes its phosphorylation by PDK1. PDK1 localizes at the plasma membrane through its COOH-terminal pH domain which binds to PIP3 and facilitates the colocalization of Akt and PDK1 3, Akt is not phosphorylated at Thr30863 linked ubiquitylation of Akt by the TNF receptor-associated factor (TRAF6) ligase also facilitates its recruitment to the plasma membrane Activation of PI 3-kinase and subsequent production of PIP308 phosphorylation In vitro studies using mutation of Ser473 to alanine has no effect on phosphorylation of AktThr308 and activation of Akt 308 phosphorylation and hence Akt activation Other mechanistic studies have probed the role of the PIF-pocket in PDK1 activity. Activation of AGC kinases that do not possess a PH domain is triggered through phosphorylation of their hydrophobic (HF) motif by mTORC1, which promotes that interaction with a motif in the PDK1 catalytic domain mTOR plays an important role in cell metabolism, proliferation, differentiation, and survival 473 takes place on the surface of the ER 308 and Ser473 have been reported as either interdependent or independent of each other 308 and Ser473, respectively. Here we hypothesize that Raptor interacts physically with PDK1 in the mTORC1 signaling complex serving as a scaffolding protein which allows PDK1 to phosphorylate Akt at Thr308. We show that PDK1 co-immunoprecipitates with Raptor, and mTOR in α2M*-stimulated prostate cancer cells. Downregulation of Raptor or PDK1 by RNAi nearly abolishes α2M* -induced phosphorylation of Akt at Thr308. Immunodepletion of PDK1 or Raptor reduced Akt at Thr308 significantly. Addition of the supernatant of Raptor or PDK1 depleted cell lysates restored AktThr308 phosphorylation. Thus, PIP3 production is a necessary, but not sufficient event in activating Akt by phosphorylation at Thr308. Whether these results will apply to activation of other PH domain-containing AGC kinases by PDK1 remains to be determined, but seems likely.Recruiting PDK1 to muscle A kinase anchoring protein (mAKABα) facilitates activation and release of the downstream target RSK 2-macroglobulin (α2M*) was prepared as described previously Thr308, p-AktSer473, Akt, and PDK1 were purchased from Cell Signaling Technology . Antibodies against Raptor were purchased from Santa Cruz Biotechnology . Anti-actin antibody was from Sigma . [33P]-γ-ATP (specific antibody 3000 Ci/mmol) was purchased from Perkin-Elmer Life Sciences . All other materials were of analytical grade and procured locally.Culture media were purchased from Invitrogen. Activated α2 incubator at 37°C. After reaching 90% confluency, the medium was aspirated and a fresh volume of RPMI-S medium and the cells were used for experiments described below.We have used two human prostate cancer cell lines 1-LN and DU-145. The highly metastatic 1-LN cell line is derived from less metastatic PC-3 cells in nude mice and was a kind gift of Dr. Philip Walther  2M*-stimulated cells. 1-LN cells were grown over night in RPMI-S media in 6 well plates (3×106 cells/well) at 37°C in a humidified CO2 (5%) incubator. At about 90% confluency the medium was aspirated, cells washed 2x with cold HHBSS, a volume of RPMPI-S added and after 5 min of temperature equilibration; cells in respective were stimulated with either buffer or α2M* (50 pM/25 min) and incubated as above. The reaction was terminated by aspirating the media, and cells lysed in a volume of CHAPS lysis buffer B containing 40 mM HEPES  was added and cells lysed over ice for 15 min. The lysates were transferred to separate Eppendorf tubes, centrifuged (1000 rpm/5 min/4°C) and supernatants used for protein estimation and immunoprecipitation. Equal amounts of lysate proteins (200–250 µg) in separate experiments were used for immunoprecipitation with Raptor antibodies . PDK1 antibodies (1∶50) and mTOR antibodies (1∶50) followed by the addition of 40 µl protein A agarose and contents incubated with rotation overnight at 4°C. Raptor, PDK1, mTOR immunoprecipitates were recovered by micro-centrifugation (2000 rpm/5 min/4°C) and washed twice with cold lysis buffer B. To each immunoprecipitate a volume of 4x sample buffer was added, tubes boiled for 5 min centrifuged, electrophoresed (4–20% or 10% acrylamide gels), transferred to Hybond-P membranes and the respective membranes immunoblotted with Raptor, PDK1, and mTOR antibodies. The protein bands on the membranes were visualized by ECF and STORM 860 Phosphorimager (GE Lifesciences). The specificity of antibodies employed here and in the experiments described below was determined by treating the cells with non-immunoantibodies and cell lysates processed as above under the experimental conditions, no reactivity of these controls was observed. The membranes were reprobed for protein-loading controls Colocalization of interacting proteins has been widely studied by co-immunoprecipitation techniques. To test the hypothesis PDK1 is an interacting protein with Raptor in the mTOR signalosome, we first studied the colocalization of PDK1 with mTOR, and Raptor by co-immunoprecipitation in α308 by PDK1, we have used RNAis to downregulate the expression of PDK1 or Raptor. The chemical synthesis purification and annealing of PDK1 dsRNA were performed by Ambione of Raptor by Sigma. The PDK1 sense sequence is 5′ - CAG AAG AUC AUU AAG UUG CAA-3′; antisense 5′ - UUA CAA CUU AAU GAU CUU CUG-3′. The Raptor sense sequence is: 5′ - CUC AAC AAA UCU UUG CAG At-3′ (ID# SAS1_HSO1_00048387); antisense 5′ - UCU GCA AAG AUU UGU UGA Gels - 3′ (ID# SAS1_Hs) 1-00048387_AS.To understand the mechanistic details of phosphorylation of Akt at Thr6 cells/well 6 well plates) were treated as follows: (1) lipofectamine + buffer; (2) lipofectamine + α2M* (50 pM/25 min); (3) respective dsRNA (100 nM/48 h) then α2M* (50 pm/25 min); or (4) scrambled dsRNA (100 nM/48 h) then α2M*. We have used insulin (200 nM/15 min) as a positive control in these studies. The reactions were terminated by aspirating the medium, a volume of CHAPS lysis buffer added, cells lysed over ice for 15 min, centrifuged (1000 pm/5 min/4°C), supernatants transferred to new tubes and protein concentration determined. As desired, equal amounts of lysate proteins (200–250 µg) were immunoprecipitated with either anti-PDK1 or Raptor antibodies as described above. Phosphorylation of Akt at Thr308 by PDK1 or Raptor immunoprecipitates was determined by kinase assays described elsewhere 2, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine and 20 µg/ml leupeptin was added followed by the addition of 30 µM AktThr308 kinase substrate (NH2-KTFCGTPEYLAPEVRR-COOH) to the respective tubes. The peptide (NH2- GGEEEELYFELVKKKK-COOH (ZAK2 peptide) served as the control. The reaction was initiated by adding 50 µM ATP and 5 µCi [33P]-γ-ATP in each tube and tubes incubated for 30 min at 30° in a shaking water bath. The reaction was stopped by the addition of 5 µl of 0.5 M EDTA in each tube. The tubes centrifuged at 3000 rpm for 3 min, 40 µl of each supernatant applied on p81 phosphocellulose paper, allowed to dry and paper washed four times each time by immersing them in a liter of 1N H3PO4 for 3 min. The papers were rinsed with acetone and that radioactivity was counted in a liquid scintillation counter. In preliminary experiments kinase activities of AktThr308 kinase towards ZAK3 peptide were always 50 to 60% of buffer control. Hence control peptide activities are not being shown.In all the experiments described below, downregulation of PDK1 or Raptor expression was achieved by transfecting the cancer cells with the respective dsRNAs (100 nM/48 h). Downregulation of PDK1 or Raptor, as measured by Western blotting, was ∼65–70% 2M/insulin stimulated AktThr308 phosphorylation by PDK1, we also studied the effect of immunodepleting Raptor and PDK1 on AktThr308 phosphorylation. The 1-LN prostate cancer cells were grown and stimulated with α2M* (50 pM/25 min) or insulin (200 nM/15 min) as described above. The reaction was terminated by expirating the media and cells lysed in lysis buffer A containing 50 mM Tris•HCl (pH 7.5) 120 mM NaCl 0.1% NP40, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 1 mM benzamidine and leupeptin (10 µg/ml) as above. The lysates were centrifugated at 1000 rpm/5 min/4°C, supernatants collected and protein concentration determined. In separate experiments to deplete PDK1 and Raptor in cell lysates, to an equal amount of lysate protein was added anti-PDK1 or Raptor antibody and immunoprecipitation of PDK1 and Raptor was done as described above. Washed PDK1 and Raptor immunoprecipitates were used for the assay of AktThr308 phosphorylation in absence or presence of cell lysates remaining after removal of PDK1 or Raptor, which was assessed by immunoblotting of PDK1 and Raptor and found to be negligible (data not shown). AktThr308 phosphorylation was assayed as described above.In addition to down regulating the expression of Raptor and PDK1 respective by RNAis on αStatistical analysis significance of the data was determined by students “t” test. P values of 0.05 are considered statistically significant.2M* causes a 2-3-fold increase in mTOR, Raptor, p-S6K, P-4EBP1, p-AktThr308, and p-AktSer473 in a time and dose-dependent manner 2M* demonstrated co-immunoprecipitation of mTOR, Raptor, and PDK1 (2M*. Since Raptor is a scaffold protein and does not have a kinase function, we suggest that PDK1 uses Raptor as a platform for phosphorylating Akt at Thr308.Stimulation of 1-LN prostate cancer cells with αand PDK1 . These s308 we silenced the expression of PDK1 or Raptor by RNAi and obtained Raptor immunoprecipitates from cells stimulated with α2M* (2M*, silencing PDK1 gene expression significantly reduced phosphorylation of Akt at Thr308 as determined by Western blotting to detect p-AktThr308 in cell lysates (Thr308 kinase. We have shown earlier that down regulation of Raptor by RNAi significantly reduced phosphorylation of S6K at Thr389 and Thr229 and 4EBP1 389 while PDK1 phosphorylates S6K at Thr229. Since down regulation of Raptor not only reduced phosphorylation of S6K at Thr389 mediated by mTORC 229 we suggest that Raptor plays a role in the kinase activity of PDK1. We thus next studied phosphorylation of Akt at Thr308 by PDK1 employing kinase assays in Raptor immunoprecipitates of cells transfected Raptor RNAi and stimulated with α2M* (2M*-induced phosphorylation of Akt at Thr308 (308 and deficiency of either suppresses phosphorylation of Akt at Thr308. Similar results were obtained on phosphorylation of Akt at Thr308 in Raptor and PDK1 immunoprecipitates of 1-LN and DU-145 prostate cancer cells transfected with PDK1 and Raptor dsRNAs (Thr308 phosphorylation in Raptor immunoprecipitates of 1-LN cells stimulated with insulin as well as α2M* (T308 phosphorylation by PDK1. Since Raptor does not possess kinase activity, Raptor and PDK1 immunoprecipitates of cell lysates should demonstrate the same ability to phosphorylate Akt at Thr308 as is found in the current study. These studies further support our hypothesis that Raptor in the mTORC1 complex is required for PDK1-mediated T-loop phosphorylation of Akt and suggest that like mTORC2, mTORC1 complex also participates in Akt activation.To further probe the proposed interaction of Raptor with PDK1 in the phosphorylation of Akt at Thrith α2M* . In prosith α2M*  and in c lysates . These sith α2M* . Silencit Thr308 . Thus cot Thr308  and Raptr dsRNAs . Insulin as α2M* . We show308 by PDK1, we immunodepleted α2M*-stimulated 1-LN cell lysates of Raptor with anti-Raptor or PDK1 antibodies. We measured phosphorylation of Akt at Thr308 by kinase assay in Raptor or PDK1 immunoprecipitate in absence or in presence of cell lysates depleted of Raptor (2M* or insulin (308 in both α2M and insulin-stimulated cells (Thr308 phosphorylation (Thr308 in both α2M* and insulin-stimulated cells (308 by PDK1 and indicate the assembly of multiprotein signaling complex in initiating PDK1 signaling network. Formation of a similar multiprotein signaling complex has recently been suggested.To further analyze the requirement of Raptor in Akt phosphorylation at Thrf Raptor  or PDK1  insulin . Raptor ed cells . Additiorylation . This ised cells . Additioed cells . These r308 and is implicated in critical cellular processes including survival, metabolism, and tumorigenesis. Activation of PDK1 in cancer through genomic or epigenetic changes is often associated with more aggressive tumors and poor prognosis.PDK1 is a serine/threonine kinase that phosphorylates members of the conserved AGC kinase family including Akt at Thr2M* binds to cell surface GRP78 and activates downstream pro-proliferative and anti-apoptosis signaling cascades including RAS/MAPK, PI 3-kinase, PDK1, Akt and mTORC1 and mTORC2 signaling 308 does not occur in either PDK1 null mice embryonic stem cells or tissue specific PDK1-knockdown mice 308 in these mice was markedly reduced Activated α513 regulates PH domain dimer-monomer equilibrium thereby converting an inactive dimer to an active monomer. Membrane bound PDK1, self-activates via autophosphorylation resulting in phosphorylation of the activation loop residue Ser241 and of PH domain residues Ser410 and Thr513. Subsequently, the active PDK1 kinase can phosphoactivate plasma membrane targets or can dissociate from membrane and phosphorylate cytoplasmic targets 308Regulation of PDK1 activity occurs by several mechanisms including post-translational phosphorylation, which induces conformational changes stabilizing the protein, serving as anchors with respect to interaction with other proteins, or regulating the intracellular targeting of PDK1 to specific compartments such as cytoplasm, plasma membrane or the nucleus 308 in mTOR immunoprecipitates of prostate cancer cells stimulated with α2M* Thr308 phosphorylation, therefore, is PIP3-dependent and should take place at the plasma membrane. We do not understand the precise mechanistic and subcellular location of Raptor-mediated phosphorylation of Akt at Thr308 reported here. PDK1 in unstimulated cells is mostly cytosolic in occurrence, but stimulation with growth factors causes translocates to plasma membranes. PDK1 also may translocate to nuclei S473 occurs on the surface of the ER 308 and Ser473 have been reported to be both interdependent or independent of each other 308 and Ser473, respectively. This view is substantiated by the presence of both p-AktThr308 and p-AktSer473 in Raptor immunoprecipitates of 1-LN cells stimulated with α2M* (308The PI 3-kinase inhibitor LY294002, inhibits phosphorylation of Akt at Thrith α2M* . We haveThr308 requires Raptor as a scaffold or co-factor in addition to the requirement for phosphoinositide participation in Akt activation. Thus PIP3 generation is a necessary, but not sufficient requirement in PDK1-dependent phosphorylation of AktThr308. A recent study suggests that physical association of PDK1 with Akt1 is sufficient for Akt phosphorylation in the T-loop, which is independent of PIP3/PIP2 induced plasma membrane translocation In conclusion, in this study we employ co-immunoprecipitation, RNAi, and immunodepletion techniques to demonstrate that PDK1-mediated phosphorylation of Akt


The bacterial communities in the unenriched waters and ice consisted of the classes Flavobacteria, α-and γ-Proteobacteria, which are frequently found in natural sea ice in polar regions. Furthermore, the results indicate that seawater bacterial communities are able to adapt rapidly to sudden environmental changes when facing considerable physicochemical stress such as the changes in temperature, salinity, nutrient status, and organic matter supply during ice formation.The structure of sea-ice bacterial communities is frequently different from that in seawater. Bacterial entrainment in sea ice has been studied with traditional microbiological, bacterial abundance, and bacterial production methods. However, the dynamics of the changes in bacterial communities during the transition from open water to frozen sea ice is largely unknown. Given previous evidence that the nutritional status of the parent water may affect bacterial communities during ice formation, bacterial succession was studied in under ice water and sea ice in two series of mesocosms: the first containing seawater from the North Sea and the second containing seawater enriched with algal-derived dissolved organic matter (DOM). The composition and dynamics of bacterial communities were investigated with terminal restriction fragment length polymorphism (T-RFLP), and cloning alongside bacterial production (thymidine and leucine uptake) and abundance measurements (measured by flow cytometry). Enriched and active sea-ice bacterial communities developed in ice formed in both unenriched and DOM-enriched seawater (0–6 days).  In addition to sea ice in the Arctic and Southern Oceans, seasonal sea ice also covers large areas of marine and brackish waters at lower latitudes, such as the Baltic Sea and the Sea of Okhotsk  compounds  thrive in nutrient-rich waters and is thought to metabolize exudates from growing phytoplankton , which was done at low light. Protists were counted by epifluorescent microscopy and they were found only in very low numbers  in Germany in October 2009. Water from the North Sea was collected near Helgoland  and transferred with a clean food-quality road tanker to the HSVA within 24 h of initial collection. The water temperature was 13°C when collected and was maintained at this temperature (±1°C) during transport. Eighteen polyethylene (PE) bags were placed into the test basin, using a random block design, and each bag was filled with 1.2 mμmol L−1 and in the NSW 109.4 and 4.18 μmol L−1, respectively. The first sampling (day 0) was performed in all 18 mesocosms 2 days after water had been pumped into the mesocosms.GF/F-filtered algal-derived organic matter  and spraying a fine mist of Milli-Q water  over the surface of the basin to ensure ice nucleation , the sea ice had grown <1 cm and the ice was too thin and unstable to perform all the measurements needed. Therefore, the ice cover was left intact, and only the under ice water was sampled from all bags through the pressure-release PVC tubes. The sea ice had consolidated by day 4 and ice and underlying waters from three replicate mesocosms were sampled from both the DOM-enriched and-unenriched treatments on days 4–6. Unfortunately on the first ice-sampling day (day 4), one of three replicates of the DOM-enriched and two of three replicates of the unenriched mesocosms were compromised and not included in the subsequent analyses. Once a mesocosm had been sampled for ice it was not sampled again, as the removal of the ice completely altered future ice growth. Brine and frost flowers were collected on the last two sampling days (days 5 and 6).Under ice water sampling was performed through the pressure-release PVC tubes, using 50-mL plastic syringes and small-bore Teflon tubes. Ice was sampled by sawing ice blocks, which were then floated in a container held beneath the ice to minimize brine drainage from the skeletal layer. The ice blocks were carefully removed and immediately cut into two or three sections depending on ice thickness: The top and middle ice sections were always 4 cm and the bottom ice varied between 3 and 8 cm. The ice sections were placed into autoclaved polypropylene (PP) buckets, and melted at room temperature (melt water not exceeding 4°C). As soon as the ice had melted the samples were transferred to a cold room (4°C) and the water was filtered within 2 h.Brines were collected on days 5 and 6 from sackholes . All filters were immediately frozen at −20°C and subsequently stored at −80°C. Organic and inorganic nutrients, carbohydrates, oxygen, temperature, and salinity data were also collected during the experiment. The methods employed are described by Aslam et al.  and stored at 4°C. The cells were stained with SYBR Green I  at a final dilution of 1:10 000 for at least 10 min in the dark and analyzed with an LSR II flow cytometer  using a 488-nm laser  were added to each sample to calculate the volume of sample used in counting. Bacterial data were typically acquired until 50,000 events were recorded. Cell populations of high-and low-nucleic acid content  bacteria were identified from bivariate plots of green fluorescence versus SSC (sideward light scatter), based on differences in green fluorescence using FACS Diva software (BD Biosciences). The cell abundance (cells per mL) for each population was calculated from the sample volume and number of recorded events identified as bacteria.IS = (Xi/Si) * (SW/XW)  seawater from the sample bags was added to the scintillation vials. All the work was done at 4°C.Bacterial production measurements were carried out to evaluate the bacterial net biomass production, based on amount of DNA and protein synthesis. The samples contained a known amount of crushed ice and sterile-filtered seawater and were processed with the following techniques, as described by Kaartokallio : In shor14C-leucine  and [methyl-3H] TdR . The concentrations used, 30 nmol L−1 for TdR  and 1200 nmol L−1 (ice samples) and 400 nmol L−1 (water and brine samples) for Leu, were verified to be above the saturating concentrations .DNA was extracted from the filters, using a PowerSoil® DNA isolation kit  according to the manufacturer's instructions with one additional step: the filters were crushed with a sterile pipette tip after they were transferred to the PowerBead tubes. The extracted DNA was used as a template (10–150 ng) to amplify the 16S rRNA genes for the terminal restriction fragment length polymorphism (T-RFLP) and clone libraries. For T-RFLP, PCR was performed with a 6-carboxyfluorescein-labeled forward primer . Taxonomic identification of the 16S rRNA genes was done, using a naïve Bayesian classifier  of the Ribosomal Database Project  obtained, one from unenriched seawater on day 0, another from unenriched bottom ice on day 5 and the third from DOM-enriched bottom ice on day 5. Cloning of the amplified 16S rRNA genes, plasmid extractions, and sequencing were performed in Helsinki University, Institute of Biotechnology. Approximately 950 bp of the 16S rRNA gene were sequenced from the 5′ terminus of the 16S rRNA gene. The sequences were corrected manually with the Staden Package 1.6.0 Gap v. 4.10 . Mothur LIBSHUFF , using the average neighbor algorithm and 10,000 iterations in Mothur v. 1.21.1  virtual digest program (Sulfolobus tokodaii (AB022438) was used as an outgroup in the alignment. A phylogenetic NJ tree was visualized with Interactive Tree Of Life  and the Kruskal–Wallis (KW) rank sum test, respectively, with a base package of R software  analysis was performed on the Bray–Curtis distance matrix derived from square root transformed relative abundance data. Square root transformation was performed in order to balance rare and abundant species, as the Bray–Curtis resemblance measure uses no form of scaling and our samples showed large differences between their relative abundances.P-values, 9999 permutations were used. All community structural analyses were performed, using PRIMER v. 6 . As there were no statistical differences, either in salinity or temperature in the ice, the differences in brine salinity were most likely caused by the sampling techniques used. The average ice thickness varied from day 4 to day 6 by 7.5 cm , 10.3 cm , and 11.2 cm , respectively.The salinities and temperatures during the experiment are shown in Table W = 275, P = 1.469 × 10−5; W = 267, P = 6.723 × 10−5; W = 259, P = 0.0002548, respectively) and water . As the number of heterotrophic nanoflagellates was low throughout the experiment, as verified by epifluorescence microscopy, bacterial abundances give an estimate close to the maximum values attainable without flagellate grazing  and high-(HNA) nucleic acid content populations and total bacterial abundances (HNA + LNA) are presented in Figure Is) , whereas significant differences were not detected in the under ice waters.On the first ice-sampling day (4), the bacterial enrichment index (n = 3), brine (n = 3) and ice (n = 1), whereas in the unenriched samples, only one ice sample contained small aggregates. In the DOM-enriched samples, the aggregates were estimated to contain on average 130–160 cells in brines and 200–240 cells in water, counting an average of 44% and 48% of all bacterial cells, respectively. In the unenriched samples, the aggregates were smaller, containing only 8–14 cells and constituting on average 11% of all bacteria. As the aggregates were detected only in one ice sample in the DOM-enriched samples and the proportion of the aggregates in the unenriched samples was very low, the bacterial cell aggregation observed cannot explain the declining abundance in the DOM-enriched ice samples. Instead, the aggregation may point out the onset of stationary growth in the experiment.We observed formation of bacterial cell aggregates (counted by epifluorescence microscopy) in both the unenriched and DOM-enriched samples on the last sampling day (day 6). In the DOM-enriched samples, the aggregates were found in water . In the total Leu incorporation , the signed-rank test used did not identify significant differences between the unenriched and DOM-enriched samples. In the unenriched water samples, both Leu and TdR increased only slightly from their initial values and reached maxima on the second ice-sampling day (5). In the DOM-enriched under ice water samples, there was a pronounced increase in TdR immediately after freezing began (day 3) that continued until the end of the experiment, whereas an increase in Leu was not detected until day 4.Salinity-normalized bacterial production measurements are presented in Figure W = 306, P = 4.408 × 10−10 for both). In brines, the Leu and TdR incorporation rates were low compared with those measured in the bulk sea ice.In the ice, the temporal succession of Leu and TdR uptake incorporation rates reflected those in the water. For ice, both rates were significantly higher in the DOM-enriched samples (P = 0.0001 with 9999 permutations for both) with all three restriction enzymes  gave the smallest cross-validation error (misclassification 5.8%) and explained 90.3% of the total variability in the T-RF data. Over time the corresponding values were m =14, misclassification 21.2%, and 96.3% explained.Generalized discriminant analysis , after which α-Proteobacteria began to dominate alongside Flavobacteria  as well as between unenriched bottom ice and DOM-enriched bottom ice (P < 0.0001). These differences suggest that DOM-enrichment induced the strongest changes in the bacterial communities. Moreover, the DOM-enriched bottom ice communities were separated into a distinct group from intermixed sequences of initial unenriched seawater and unenriched bottom ice communities in the phylogenetic NJ tree , species evenness between the unenriched and DOM-enriched bottom ice was notably reduced.The OTU numbers observed in the clone libraries began to reach saturation at an evolutionary distance of 0.15, except in the initial unenriched water sample . Both species richness and evenness were reduced after freezing, with the indices being lowest in the DOM-enriched samples Table . As the In both unenriched and DOM-enriched North Sea water treatments, the bacteria abundance in newly formed sea ice was higher than in initial water samples based on the enrichment index. Salinity-normalized bacterial abundance was highest in the frost flowers, exceeding that in the sea ice and brine, as was also found in previous study (Bowman and Deming 4+) and particulate organic carbon , and decrease in DOC together with an increase in ammonium  and DOC consumption in these samples. Both Shewanella sp. and Colwellia sp. have been found in natural sea ice  in the initial water of the unenriched samples compared to the DOM-enriched samples. The sudden decrease in γ-Proteobacteria in the unenriched samples could also have been due to viral attack, as very high virus-to-bacteria ratios have been reported in Arctic sea ice , benefit from high concentrations of LMW compounds (Teira et al. γ-Proteobacteria suggests that they can effectively outcompete α-Proteobacteria under high nutrient concentrations, as previously suggested by Pinhassi and Berman (γ-Proteobacteria also seems possible in sea-ice under elevated concentrations of labile substrates. Conversely, the increase in Flavobacteria in the unenriched samples suggests their superior competitiveness over α-and γ-Proteobacteria in sea ice under lower nutrient concentrations.With the exception of the transient d Berman . Similard Berman , the oppAn active sea-ice bacterial community, similar to those previously described in natural sea ice, developed in sea ice experimentally grown from temperate North Sea water that does not typically freeze and produce sea ice.α-proteobacteria, and γ-proteobacteria, whereas the DOM-enriched community was taken over by γ-Proteobacteria, indicating their opportunistic growth due to the elevated concentrations of available DOM. In all, the results show that seawater bacterial communities have a capacity to rapidly adapt when facing considerable changes in temperature, salinity, or supply of nutrients and organic matter occurring e.g., during ice formation.Bacterial abundances relative to salinity were notably higher in ice compared to initial seawater in both unenriched and DOM-enriched treatments despite the absence of algae and other protists. Bacterial production and the changes in bacterial communities were pronounced in the DOM-enriched experimental units, pointing to the importance of substrate supply as a regulator of bacterial growth in sea ice and underlying waters. The bacterial communities in the unenriched samples were not different from those in the initial seawater, whereas the bacterial communities in the DOM-enriched samples were significantly different from the communities in initial seawater and unenriched samples. The unenriched sea-ice communities were dominated by Flavobacteria,


Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase  and protein kinase C that are often deregulated in cancer and are important therapeutic targets. The ability of cells to evade cell death can lead to cancer. Programmed cell death is a complex series of well-orchestrated events that result in the controlled demise of the cell. Apoptosis or type I programmed cell death is characterized by the activation of proteases that results in the destruction of cellular components and cell shrinkage facilitating its phagocytosis. Regulated necrosis or type III programmed cell death is a series of ordered events that lead to a more explosive form of death .Autophagy, a process of “self-eating”, is often activated in response to starvation and stress . SeveralAutophagy has been regarded as a survival mechanism since it provides nutrient deprived cells with a means of survival. However, autophagy has been regularly observed in dying cells . This raThe process of autophagy involves a series of steps, including induction, cargo recognition and selection, vesicle formation, autophagosome-lysosome fusion, digestion of cargo and finally release of digestion products into the cytosol . AutophaBeclin1, and Atg7 are deleted in several cancers [Several studies point to the tumor suppressive functions of autophagy. Beclin 1 is a haploinsufficient tumor suppressor ,29. It h cancers . Moreove cancers . Also, t cancers . However cancers , mediate cancers  and resi cancers . It is b cancers . Thus, a cancers .Given the ambiguous role of autophagy in cancer, there is a growing interest in the pathways and molecules that regulate it. Kinases play an integral role in the inception and execution of autophagy. While some kinases such as mTOR, PI3K and AMPK directly regulate components of autophagic machinery, the precise role of other kinases such as mitogen-activated protein kinase (MAPK) and protein kinase C in autophagy is less well characterized and they regulate autophagy indirectly by the modulation of the levels/function of autophagy related proteins. The purpose of this review article is to discuss current literature on the role of several kinases in autophagy regulation that has important implication in cancer and other diseases.2.The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphatidylinositol kinase-related kinase (PIKK) family . It was et al demonstrated that in the Drosophila fat body, starvation induces autophagy that peaks within hours following nutrient withdrawal and then reaches low levels over extended periods of starvation so as to promote only survival and avoid autophagic cell death [mTORC1 activity depends on the nutrient status of the cells. A direct role for mTORC1 in autophagy came from the studies that showed that mTORC1 phosphorylates and regulates proteins involved in autophagosome formation. In yeasts, TOR inhibits the association between ATG1 (ULK1 in humans) and ATG13 by hyperphosphorylating ATG13, thereby decreasing its affinity for ATG1 ,48. Recell death . The stull death  and S6K ll death .The mechanism by which S6K promotes autophagy may involve synthesis of proteins that participate in autophagosome formation and maturation ,59 or moAnother downstream target of mTOR involved in regulating excessive autophagy is death-associated protein 1 (DAP1) . DAP1 is3.As part of the energy-sensing cascade, mTORC1 is also regulated by the AMP-dependent protein kinase (AMPK). AMPK is activated in response to stress and low cellular ATP levels and phosphorylates TSC2 (tuberous sclerosis complex-2) and raptor, resulting in inhibition of mTORC1 activity ,71. Thus4.The phosphoinositide 3-kinases are enzymes that catalyze the phosphorylation of the 3′ position hydroxyl group of the inositol ring in phosphatidylinositol (PtdIns). Based on structure, function and substrate specificity, the PI3Ks have been classified into three classes . Class IThe class III PI3K, Vps34 plays an important role in mediating autophagosome formation. It directly interacts with beclin 1  and promWhile class III PI3K directly participates in autophagy, class I PI3K can regulate autophagy indirectly via Akt and mTORC1. It mediates growth factor and insulin signaling and leads to the activation of Akt via PDK1. Once activated, Akt phosphorylates and inhibits TSC2, which acts as a GTPase-activating protein (GAP) for ras homolog enriched in brain (Rheb), a GTP binding protein. GTP-bound Rheb leads to the activation of and signaling through the mTORC1 complex resulting in protein synthesis, cell growth and inhibition of autophagy . Thus, thVps34 has also been shown to positively regulate the activity of mTORC1 in response to amino acids . Amino aet al. [The observation that hVps34 positively regulates autophagosome formation as well as mTORC1, a negative regulator of autophagy, is counterintuitive. An explanation for this paradoxical role is that its binding partners such as beclin 1 and its localization determine specific functions in response to starvation or amino acids. For example, Kihara et al.  demonstr5.Mitogen activated protein kinases (MAPKs) are serine/threonine kinases that mediate responses to various extracellular stimuli. Extracellular signals such as growth factors lead to a sequential phosphorylation cascade that ultimately results in MAPK activation. Once activated, MAPKs lead to phosphorylation-dependent activation of other kinases and transcription factors. The four categories of MAPKs are extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), p38, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and big MAP kinase (BMK) . The bes5.1.ERK1 (p44) and ERK2 (p42) are two isoforms of ERK that are activated downstream of Ras in response to extracellular cues. Ras binds to and activates Raf and initiates activation of MAPK signaling cascade. Raf activates MEK which in turn phosphorylates and activates ERK1/2 . ERK conOne of the earliest studies used pharmacological inhibitors of ERK to demonstrate that it mediates starvation-induced autophagy by phosphorylation and consequent activation of GAIP (Gα interacting protein) in human colon cancer cells . ERK hasRecent studies suggest that ERK regulates the maturation of autophagic vacuoles. Activation of ERK has been associated with the formation of large cytoplasmic vacuoles -106. LinImportant insights into the role of ERK in autophagy/mitophagy came from studies in neurodegenerative disorders -112. ERK5.2.p38 MAPK family comprises of four isoforms p38α, -β, -γ and -δ . p38α isIn yeast, the p38 homolog Hog1 was shown to positively regulate autophagy in response to osmotic stress ,118 and p38 was also shown to negatively regulate autophagy. Inhibition of p38 in colon cancer cells  and myel5.3.JNK family is comprised of three members JNK1, -2 and -3 ,115. Whivs. apoptosis [JNK has been implicated in the induction of autophagy by various stimuli, including starvation , cytokinpoptosis .Besides its role in posttranslational modification of proteins that regulate autophagy, JNK can also regulate the expression of beclin 1 transcript via phosphorylation of c-Jun ,148,149.JNK can also promote autophagy via p53 in several cell types, including human colon carcinoma and fibrosarcoma ,153,154.JNK 1, JNK2 and JNK3 in neurons increased autophagy [JNK3 alone is sufficient to increase autophagy to provide neuroprotection. Moreover, future studies should determine if different isoforms of JNK have distinct role in cancer versus neurodegenerative disorders.In contrast to the above mentioned pro-autophagic role of JNK, a recent study demonstrated that targeted deletion of utophagy . The inc6.2+ and diacylglycerol (DAG) for their activity, novel PKCs are Ca2+-independent but DAG-dependent whereas atypical PKCs are independent of both Ca2+and DAG. Conventional and novel PKCs are the receptors for tumor-promoting phorbol esters, which are potent activators of PKCs and can substitute for the physiological activator DAG [Protein kinase C (PKC) is a family of phospholipid-dependent serine/threonine kinases that regulate diverse cellular functions. Based on the structural features and cofactor requirements, PKC members are classified as conventional , novel  and atypical (PKCζ and -ι) PKCs . While cator DAG . PKCs haator DAG ,161. Theator DAG ,163. Subator DAG -166. In ator DAG ,164-169.et al. [As with apoptosis, PKCδ is the first PKC isozyme shown to play an important role in autophagy. PKCδ suppressed autophagy in pancreatic ductal carcinoma cells by increasing the expression of tissue transglutaminase 2 (TG2)  which neet al.  who repoet al. . Safingoet al. . Thus, w2+ and phosphorylation of PKCs primes them for activation, it is conceivable that an alteration in PKCθ localization is responsible for ER stress-induced autophagy. Recently, it has been reported that PKC can inhibit autophagy induced by starvation or rapamycin in a PI3K-independent manner [PKCθ, another member of the novel PKC family, was shown to influence endoplasmic reticulum (ER) stress-induced autophagy but had no effect on amino acid starvation-induced autophagy . ER stret manner . Several2  by phosphatases or masking the lipid with PH domain led to mitochondrial fission and mitophagy in different cell lines [2 was associated with an increase in starvation-induced autophagy. However, co-expression of mitochondrially targeted PKCα could rescue cells from mitophagy and cell death caused by PtdInsP2 unavailability [Conventional PKCα has also been implicated in autophagy. Dephosphorylation of PtdInsPll lines . Additiolability . Previoulability . Thus, Plability . PKCα kiIn addition to the involvement of specific PKCs in autophagy, several studies have employed pharmacological activators or inhibitors of PKCs to establish their role in autophagy. PKC was shown to positively regulate oridonin-induced autophagy in HeLa cells via Raf1/JNKpathway  and acad7.2+/calmodulin-dependent kinase kinase- beta (CaMKKβ), death associated protein kinase (DAPK) and PERK have been associated with ER stress-induced autophagy. Once activated, these kinases can ultimately lead to the upregulation of autophagy-related genes and inhibition of autophagy suppressors.Autophagy is activated in response to multiple stresses such as pathogen infection, nutrient deprivation, hypoxia and endoplasmic reticulum (ER) stress . SeveralOne of the first proteins activated in response to UPR is the inositol-requiring enzyme 1 (IRE1) . In yeas2+ stored in the ER to the cytoplasm, causing activation of various Ca2+-dependent kinases. Studies that employed RNA interference and pharmacological inhibitors showed that Ca2+-induced autophagy was dependent on the Ca2+/calmodulin-dependent kinase kinase-beta (CaMKKβ), which is an upstream activator of AMPK resulting in mTORC1 inhibition and autophagy induction [As a part of UPR, PERK directly phosphorylates eukaryotic initiation factor 2α (eIF2α) to block translation initiation. Recently, it has been shown that PERK-mediated phosphorylation of eIF2α is also required for LC3 conversion during ER stress-induced autophagy ,190. It nduction ,193.2+/calmodulin-regulated kinase that plays an important role in ER stress-induced autophagy. Recently, Zalckvar et al. showed that DAPK1 mediates induction of autophagy by phosphorylating beclin 1 [L and mediates autophagosome formation [Death-associated protein kinase 1 (DAPK1) is another Cabeclin 1 . Phosphoormation . DAPK1 aormation ,196.8.The phenomenon of autophagy was described nearly half a century ago. However, research into the molecular mechanisms and regulation of autophagy has taken serious form only during the past decade. There is a growing interest in modulating autophagy for cancer therapy. While excessive autophagy is recognized as a type of cell death, its role in protecting cancer cells from stress leads to the debate as to whether autophagy should be inhibited or activated for cancer therapy. Addressing this question would require a thorough understanding of the regulation of autophagy so that appropriate proteins are chosen as targets.Posttranslational modifications of a protein often determine its function. It is now evident that autophagy can be regulated by kinases at multiple steps such as autophagosome inception, vesicle maturation, termination and autophagy-related gene expression as depicted in


T55α) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes.Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2Acis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2AD) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau.We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl T55α-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2AT55α may contribute to the hyperphosphorylation of Tau observed in AD neurons.Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A Phosphorylation/dephosphorylation of the neuronal microtubule associated protein Tau regulates in a complex manner its capacity to assemble tubulin into microtubules. It is also associated with the presence of pathological fibrils in neurons of AD patients, which are mainly composed of hyperphosphorylated Tau. Monoclonal antibodies such as AT180 and AT8, recognizing respectively the pT231 The phosphorylation of Tau is a reversible process, which implies that the pathological hyperphosphorylation can result from a deregulation of kinase and/or phosphatase activity. Intense research has increased our understanding of the kinases that generate certain phosphorylation events, as well as their complex regulation. Calpain cleavage of the regulatory p35 subunit of CDK5 results in a membrane detachment of the resulting CDK5/p25 complex D) constituted of a catalytic subunit (C subunit) and a scaffolding subunit (A subunit). This heterodimer (PP2AD) can further integrate a regulatory B-type subunit to form a heterotrimeric PP2A complex (PP2AT) T55α) has recently been determined One major brain phosphatase that dephosphorylates phospho-Tau is the Protein Phosphatase 2A (PP2A) cis/trans isomerase that targets specifically the pS/pT-P motifs. This isomerase was shown to stimulate the dephosphorylation of Tau by PP2A −/− mice show indeed a decreased phosphatase activity towards pS/pT-P motifs and an accumulation of both the pS202/pT205 AT8 and pT231/pS235 AT180 epitopes The regulation of PP2A is complex, and involves more than the subunits of the holoenzyme. Polycations can stimulate the activity of PP2A through a direct interaction with the enzyme. As the stimulation is dependent on the type of holoenzyme, the interaction involves the regulatory B-type subunit in vivo control the final phosphorylation level of Tau in vitro phosphorylated by the activated CDK2/CycA3 complex to study the effect of PP2A on its dephosphorylation. We showed previously that CDK2/CycA3 action at the AT180 epitope is equivalent to that of the combined CDK5/p25 and GSK3β kinases and can generate in a robust manner the pS202/pT205 AT8 and pT231/pS235 AT180 epitopes on Tau T55α activity towards the Tau pS202/pT205 AT8 site by the phosphorylation status of T231, whereby phosphorylation of the latter T231 site negatively interferes with the PP2A catalyzed dephosphorylation of pS202. In addition, we find that Pin1 releases this negative feedback between the pT231 and pS202/pT205 AT8 epitopes. Our findings define an additional level of molecular regulation of the PP2A phosphatase towards the phosphorylated Tau protein, whereby the interplay between kinase and phosphatase activity can potentially lead to a stable hyperphosphorylated state of Tau that characterizes AD affected neurons.Kinases and phosphatases can hence regulate the phosphorylation/dephosphorylation of Tau in a complex and concerted manner, whereby this intricate feedback can Fig. S1), recognized respectively by the diagnostic AT8 T55α complex composed of the scaffolding A subunit, the catalytic C subunit and the regulatory PR55/Bα subunit, was added to this CDK2/CycA3-phosphorylated Tau441 sample (phospho-Tau) directly in the NMR tube  yield snapshots of the dephosphorylation reaction with a one hour time resolution, corresponding to the acquisition time of a single 2D spectrum (Data S1). Integration of a peak surface for a given resonance in each 2D spectrum during this in-spectrometer dephosphorylation reaction gives information on the kinetics of the modification of the corresponding amino acid  of PP2AD heterodimer and PP2AT55α heterotrimer as measured with phosphorylase a as substrate, the dephosphorylation rate at all phospho-sites of Tau was slower for the PP2AD dimer . This stimulatory effect of Pin1 is however selective, as the dephosphorylation of pT231, monitored by the appearance of the R230(T231) peak, is still negligible.Using a cdc2 phosphorylated Tau as substrate, Zhou et al. described a stimulatory effect of Pin1 on the dephosphorylation activity of PP2AD in the absence or presence of Pin1. Although already of reduced activity when compared to PP2AT55α (compare panels C and D), the efficiency of the core enzyme slowed down even further upon the addition of Pin1 . We finally tested the effect of Pin1 on the enzymatic efficiency of the PP2AT55α phosphatase complex towards the phospho-Tau T231A mutant. The dephoshorylation of the complete AT8 epitope was faster than in the wild type phospho-Tau (compare panels A and D), but was not further stimulated by Pin1 (compare panels C and D).We next monitored the dephosphorylation of the same phospho-Tau by PP2AT55α is associated with the microtubules Tau phosphorylation is a reversible process that regulates in a complex manner its physiological role of microtubule stabilization. It is also linked to its pathological role, as the neurofibrillary tangles found inside the neurons of AD patients invariably contain a hyperphosphorylated form of Tau. Analysis with specific antibodies have shown that the latter form is characterized by the simultaneous presence of multiple phosphorylated residues, including the S202/T205 and T231 positions that constitute respectively the AT8 and AT180 epitopes T55α, the major brain isoform of PP2A directly associated to microtubules and Tau in vitro set-up with CDK2/CycA3 kinase generating the AT8 and AT180 epitopes on recombinant 15N-labelled Tau T55α selectively targets certain Tau phosphorylation sites, with fast dephosphorylation of the pT205 and pT153 sites but hardly any for pT231  isoform has a lower catalytic efficiency on all Tau phospho-sites compared to the heterotrimeric PP2AT55α, emphasizing the crucial role of the B subunit in the function of the PP2A holoenzyme. A correlation between the interaction of Tau with the PR55/Bα subunit and the catalytic efficiency of PP2AT55α was previously described T55α holoenzyme 224KKVAVVRTPPKSP236 Tau peptide that can compete with full-length Tau for binding to PP2A 66SGDD69 in order to efficiently interact with the PR55/Bδ subunit of the Xenopus mitotic PP2A heterotrimer T55α phosphatase activity. This potentially can lead to a stable state with both epitopes phosphorylated The dimeric PP2Acis/trans isomerase Pin1 T55α catalyzed dephosphorylation of wild type phospho-Tau and its absence in the Tau T231A mutant. Stimulation was most striking for the pS202 site, switching from no dephosphorylation with one unit of PP2AT55α in the absence of Pin1 to complete dephosphorylation in its presence. The effect is however not homogeneous, with at best a weak activity of PP2AT55α towards pT231 despite the presence of Pin1. Moreover, the stimulatory effect directly depends on the PR55/Bα subunit, as Pin1 rather hinders than stimulates the activity of the dimeric core enzyme. Pin1 hence counterbalances the negative regulation of PP2AT55α activity towards the AT8 site following phosphorylation of the AT80 pT231 site, in agreement with the inverse correlation between Pin1 expression and actual neurofibrillary degeneration in AD T55α has been shown to regulate the phosphorylation status of Tau in vivo, a potential trigger for PHF formation might be a shift of the balance between neuronal kinases and phosphatases. Our results show that phosphorylation of T231 directly influences the dephosphorylation rate of pS202, without necessitating an alteration in the PP2AT55α pool. We have reconstituted here the complex system of phosphorylated Tau and the trimeric PP2AT55α phosphatase in the NMR tube with several recombinant proteins, and thereby unravelled a subtle regulatory mechanism. An additional level of regulation has been shown by the prolyl cis/trans isomerase Pin1. The phosphorylation/dephosphorylation reactions in vivo might even be more complex, because of the presence of tubulin and/or other regulatory factors, but even these can be added in the NMR tube Xenopus ovocytes is feasible An additional level of regulation has been described for the phospho-dependent prolyl E. coli strain Preparation of the recombinant Tau proteins, isotopic enrichment and phosphorylation are described in −1cm−1. The complex was kept frozen at −80°C in this buffer until used.CDK2/CycA3 were prepared as described in D and PP2AT55α from rabbit skeletal muscle is described in a32P-phosphate at 30°C.Purification of PP2ANMR buffer was 25 mM Tris-d11, 25 mM NaCl, 2.5 mM EDTA, 1 mM DTT, pH 6.8. Dephosphorylation of 20 µM of CDK2/CycA3 phospho-Tau (or Tau mutants), as defined by absorption at 280 nm, was performed in a volume of 160 µl in 3 mm NMR tubes. Samples with Pin1 are 20 µM phospho-Tau/60 µM Pin1. The PP2A enzyme diluted in 20 µl buffer was directly added in the NMR tube to 140 µl of the phospho-Tau, immediately before starting acquisition of the 2D spectra series.1H,15N]-HSQC (HeteroNuclear Single Quantum Spectroscopy) were 32 scans, 2048 points in the direct dimension and 128 points in the indirect dimension. An optimized d1 of 0.8 s was used. The -HSQC are named 2D spectra in the text.Spectra were acquired at 293K on a 800 MHz equipped with a 3 mm Probe. Parameters of the [Data S1) were normalized against an invariant and intense peak in the spectrum corresponding to A85, set to 1 in the integration. Integration was performed with the Topspin 2.1 software . Integral data were fitted with a mono-exponential function.The peak integrals  and of the CDK2/CycA3 phospho-Tau protein (red). Inset: the enlarged region of the spectrum illustrates phosphorylation of residues S202 and T205.(PDF)Click here for additional data file.Figure S2Regulation of the dephosphorylation of the pS202/pT205 AT8 epitope. Details of the 2D spectrum (in red) of CDK2/CycA3 phospho-Tau after 16 hours of incubation at 25°C (293K) with PP2AD (3.5 U) superimposed on the spectrum of the unmodified Tau protein (in gray). A dephosphorylation without Pin1 and B in presence of an excess of Pin1.(PDF)Click here for additional data file.Data S1Step by step procedure of NMR data treatment.I. A serie of 2D spectra is acquired, in this example we limited the serie to the first five 2D spectra. The first one in the serie is the control experiment without enzyme and is time zero or starting point in the in spectrometer kinetics of dephosphorylation. The following 2D spectra, after the addition of the enzyme, are acquired successively while the NMR tube remains in the spectrometer. This experimental dataset corresponds to the one presented in II. Each 2D spectrum required 63 minutes for acquisition. Each data-point is thus obtained every 63 minutes: that is the time resolution in this experiment. III. The succession of the 2D spectra will allow to follow the kinetics of the reaction. Duration of each spectrum is cumulated. In this example, we cover with the five 2D spectra 252 minutes of the enzymatic reaction. IV. The surface of a peak in a boxed area in the 2D spectra (see point I) or Integrals is calculated by TOPSPIN2.1 Bruker software. This value is directly related to the amount of the corresponding amino acid residue in solution. We follow the evolution of the peak surface or Integral for each amino acid residue of interest (I1 to I4) during the reaction, as detected in each successive spectrum. In our example, I1 corresponds to pT153, I2 to pT231 and I3 to A152. I4 is linked to the resonance of amino acid residue A77. V. Data of integration is normalized against integral value of A77 (I4), an invariant and intense peak in the spectra, in order to compensate for any variation of intensity due to potential spectrum to spectrum modification of conditions . VI. Data are normalized against the highest integral value of the kinetics for each amino acid residue. In that way, the starting point or ending point of the kinetics is set to one, allowing easier comparison of the evolution of the amount of various amino acid residues during the course of the enzymatic reaction. VII. A final table is obtained containing the normalized integral values for each time-point in the kinetics. VIII. For each amino acid residue, an exponential curve is fitted to the experimental data by adjusting the value T in the exponential formula e(-t/T). The value T is found by minimizing the sum of the square differences between the calculated and observed data . This constant characterizes the rate of dephosphorylation and is reported in left column for pT153  and A152 . IX. Data-points from table in VII are graphically represented with the axis being the time-points (each 2D spectrum) and the ordinate the normalized integral value for each time-point (spectrum) reported for several amino acid residue of interest. In this example, pT153 or I1 (open squares) shows a decrease of its initial integral value or peak surface that can be interpreted as a decrease of the corresponding chemical species in solution, in this case due to the dephosphorylation by PP2A. The resonance of A152 (black dots) shows accordingly an increase of its integral value. pT231 integral value remains unaffected throughout the in spectrometer enzymatic reaction (stars). The fitted exponential curves are represented by lines.(PDF)Click here for additional data file.


In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R Integrins are α and β heterodimeric receptors required for numerous essential biological processes The active, oligomerized receptors can also initiate and propagate integrin-mediated outside-in signaling In 2005, we reported that αIIb-mediated outside-in signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the β3 cytoplasmic tail (β3Δ724) In previous work, we showed that the treatment of human β3Δ724 platelets in presence of fibrinogen with LIBS-specific mAb D3 and PT25-2, respectively caused aggregation, TxA2 production and granule secretion As we reported previously Metabolic inhibitors provided new insight into αIIb outside-in signaling. The Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin eliminated shape change, and biphasic aggregation and therefore  can block αIIb-mediated platelet aggregation, TxA2 production and granule secretion in response to stimulation by D3 plus Fg without preventing activation of αIIbβ3 Tp deficient mouse platelets to block the amplification of signaling induced by TxA2, and used β3 deficient mouse platelets to investigate the function of αIIbβ3 in PAR4 mediated platelet activation. As shown in Tp−/− platelets neither aggregated as extensively, nor phosphorylated Akt as extensively as occurred in wild type platelets, in response to stimulation by AYPGKF. Also, apyrase totally inhibited Tp−/− platelet aggregation and Akt phosphorylation induced by AYPGKF. These results implied that PAR4 mediated platelet activation and Akt phosphorylation are amplification dependent. However, exogenous Fg eliminated the inhibitory effects of apyrase. Therefore, α-granule secretion is ADP-dependent, and Akt phosphorylation caused by AYPGKF apparently is aggregation dependent. This conclusion was confirmed by the ability of the mAb 1B5 (prevents Fg binding to mouse αIIbβ3) to prevent platelet aggregation and Akt phosphorylation in platelets stimulated by the PAR4 agonist peptide in the presence of Fg (3 deficient platelets (which cannot aggregate) stimulated with AYPGKF to phosphorylate Akt ], or αIIbβ3-Δ724 were constructed. These stable CHO cell lines were evaluated for their ability to spread on immobilized Fg. As with CHO cells transfected with control (no insert) vectors, CHO cells expressing αIIbβ3-Δ724 did not spread on immobilized Fg, while CHO cells expressing normal αIIbβ3 (designated β3) or αIIbβ3-scramble spread fully on immobilized Fg (724KEFAKFEEER734 is not required for spreading because CHO cells expressing αIIbβ3-scramble (E724AERKFERKFE734) rather than the normal β3 sequence R724KEFAKFEEER734, spread normally. Spreading by CHO cells expressing normal αIIbβ3 supports the conclusion that αIIb outside-in signaling is not required for the spreading of CHO cells expressing normal αIIbβ3 on immobilized Fg. Apparently as with platelets The palmitoylated peptide p-RKEFAKFEEER blocked secretion and Akt phosphorylation in human Δ724 platelets treated with D3 plus Fg, and in wild type murine platelets stimulated by the PAR4 agonist peptide. These results confirm our previous observation that the β3 Rlized Fg . These r724KEFAKFEEER734. In view of the results obtained with the modified CHO cells in suspension, it cannot reasonably be argued that the inhibitory results obtained by treating AYPGKF stimulated wild type mouse platelets with p-RKEFAKFEEER were the result of interference of the palmitoylated peptide with β3-mediated inside-out or outside-in signaling. This follows from the fact that replacing the normal β3 sequence R724KEFAKFEEER734 with a scrambled version of that sequence (E724AERKFERKFE734) eliminated the ability of the cytoplasmic domain of β3 to prevent Akt phosphorylation.Akt phosphorylation was also investigated in all these cell lines in suspension, treated with D3 plus Fg. There was no Akt phosphorylation in the absence of stimulation (resting condition) . After s724KEFAKFEEER734Bidirectional αIIbβ3 mediated signaling plays an essential role in hemostasis and thrombosis. Contrary to β3-mediated bidirectional signaling, αIIb mediated signaling has not been investigated in detail. Our previous work documented that either of two LIBS-specific mAbs (D3 and PT25-2) plus Fg  and the PI3K/Akt pathway is unknown. Likewise, the identity of the SFK(s) activated by αIIb signaling is unknown. Also, the mechanism of negative regulation of αIIb-mediated outside-in signaling by key amino acids in β3 R724KEFAKFEEER734 prevents activation of the PI3K/Akt pathway. Therefore, our results identified some of the components used by platelets to propagate αIIb-mediated outside-in signaling, an important aspect of platelet function that has not been characterized in detail.In summary, we found that SFK(s) elicited PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets. Also, our data demonstrate that the negative regulation of αIIb-mediated outside-in signaling by the β3 cytoplasmic domain residues RAfter written, informed consent was obtained, and as part of a routine health exam, extra blood was collected for research purposes from the variant thrombasthenia patient. Approval for the provision of blood for research purposes was obtained from the Institutional Review Board of the Cincinnati Children’s Hospital. After written informed consent, blood was also obtained from a healthy donor to serve as a source of control platelets. The animal research was approved by the Shanghai Jiao Tong University School of Medicine Animal Care and Use Committee (Approve No. SYXK2008-0050).PP2, wortmannin, and U0126 were from Calbiochem . Apyrase, PGE1, fibrinogen (Fg) were from Sigma-Aldrich . The anti-phospho-Akt (Ser473), and the anti-actin antibodies were from Cell Signaling Technology . The PAR4 agonist peptide, AYPGKF was a generous gift from Dr. Zhongren Ding . The monoclonal antibody (mAbs) D3 Tp−/−) mice were derived as described β3 deficient and littermate control mice.The TxA2 receptor deficient . Aggregation was measured in the Lumi-Aggregometer  using washed platelets (300 µl) adjusted to approximately 106 platelets / µl with stirring at 1000 rpm.Mice were anesthetized by 1% pentobarbital sodium. Blood was collected from the abdominal aorta of the mice and anticoagulated with White’s anticoagulant  The patient has a variant form of Glanzmann thrombasthenia designated here as β3Δ724 6 platelets / µl with stirring at 1000 rpm.After informed consent was obtained, blood was collected from healthy donors and the variant thrombasthenia patient into empty syringes and then transferred to polypropylene centrifuge tubes containing 100 µl/mL  Whites anticoagulant , 0.1 µg/mL PGE1, and 1 U/mL apyrase. Platelet-rich plasma (PRP) was prepared by differential centrifugation. Washed platelets were prepared as described ATP secretion was evaluated using the CHRONO-LUME reagent  as described TxB2, a stable metabolite of TxA2, was measured as described PF4 (platelet factor 4) is released from the α-granules by the activated platelets. The supernatant fractions of platelet suspensions were collected and assayed using Asserachrom PF4 quantitative enzyme-linked immunosorbent assay (ELISA) kit  as described 5′-CCGCTCGAGGCGGGAGGCGGACGAGAT-3′ (containing Xho I restriction enzyme site), and the following downstream primers: 5′-ACGCTCTAGAGGGAGG GGAAAGGAGGCA-3′ (β3), 5′-CTAGTCTAGACTAGTCGTGGATGGTGATGAGGA-3′ (Δ724) containing Xba I restriction enzyme cutting site. The resultant PCR products were cloned into pLVX-IRES-Puro vector (Clontech). pLVX-IRES-Puro-scramble  was generated by overlap extension PCR using flanking primers: 5′-CCGCTCGAGGCGGGAGGCGGACGAGAT-3′  and 5′-ACGCTCTAGAGGGAGGGGAAAGGAGGCA-3′ , and the following overlapping mutagenesis oligonucleotides: 5′-ATCACCATCCACGACGAAGCTGAGCGAAAATTCGAACGCAAATTTGAAGCCAGAGCAAAATGG-3′, and 5′-CCATTTTGCTCTGGCTTCAAATTTGCGTTCG AATTTTCGCTCAGCTTCGTCGTGGATGGTGAT-3′. pLVX-IRES-ZsGreen-αIIb (full length of human αIIb) was generated from human platelet cDNA by PCR using the following primers: 5′-CCGGAATTCAGATGGCCAGAGCTTTGTGT-3′ (upstream primer), and 5′-CTAGTCTAGATGTAGGCTGCACCATCACTC-3′ (downstream primer). The resultant PCR product was cloned into pLVX-IRES-ZsGreen (Clontech).pLVX-IRES-Puro-β3 (full-length human β3), pLVX-IRES-Puro-Δ724 (β3 with amino acids 1–724) were generated from human platelet cDNA by polymerase chain reaction (PCR) using a common upstream primer: Lenti-X cell (Clontech) was used as the lentivirus packaging cell. Lentiviruses were produced by co-transfecting Lenti-X cells with pLVX-IRES-ZsGreen/pLVX-IRES-puro, pspAx2 and pMD2G at 3∶2:1 proportion. The lentiviruses were harvested after transfection, and depletion of the cell debris by centrifugation. CHO cells (Chinese Hamster Ovary cells) were infected with a lentivirus mixture of blank vectors, αIIb and β3, αIIb and β3-scramble, αIIb and β3-Δ724, respectively. Forty eight hours after infection, the infected CHO cells were cultured for two weeks in selective medium containing puromycin (7.5 µg/ml). The viable, GFP positive CHO cells were sorted by FACS. Then the sorted CHO cells were cultured in medium containing puromycin (5 µg/ml) for two weeks. This procedure resulted in the isolation of stable CHO cell lines expressing blank vectors, αIIbβ3, αIIbβ3-scramble, or αIIbβ3-Δ724, respectively. Then, these cell lines were cultured in medium without serum for 12 hrs before further experiments.The CHO cell lines were allowed to spread for 2 hrs at 37°C on chamber slides coated with 100 µg/ml Fg. Adherent cells were rinsed with PBS and fixed with 4% paraformaldehyde at room temperature. Then the cells were washed with labeling buffer  and incubated with Rhodamine-phalloidin for 30 min at room temperature for F-actin staining. The slides were washed twice with PBS for the fluorescence microscopy survey. Cell spreading size was quantified using National Institutes of Health (NIH) ImageJ. Statistical significance between groups was determined using a Student t test.2+, stimulated with D3 (5 µg/ml), Fg (100 µg/ml) at 37°C for 5 min with stirring at 1000 rpm. Lysates of CHO cells were prepared by adding an equal volume of 2× SDS loading buffer to each cell suspension.For D3 plus Fg stimulation, the cells were detached from the plates, and resuspended in modified Tyrode solution containing 2 mM CaEach platelet and CHO cell lysate was boiled at 100°C for 10 min, then resolved on a sodium dodecyl sulfate (SDS), 10% polyacrylamide gel and then transferred to PVDF membrane. Western blots were performed using anti-phospho-Akt (Ser473), or an anti-β-actin antibody at a 1∶1000 dilution, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody at a 1∶5000 dilution. Blots were developed using Supersignal chemiluminescent substrate .Figure S1PMA induced spreading of CHO cells expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724. (A) In presence of 100 ng/ml PMA, spreading of CHO cells expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724, respectively on immobilized Fg for 90 minutes. (B) Quantification of area (pixel number) in 4 random fields (mean ± SEM) all at 90 minutes. The size of CHO cells, CHO cells expressing αIIbβ3-WT, αIIbβ3-scramble and αIIbβ3-Δ724, was 22519.38±4321.64 pixels, 148891.15±28420.39 pixels, 121496.23±12324.66 pixels, 75023.19±11098.12 pixels, respectively. Statistical analysis performed using Student t test.(TIF)Click here for additional data file.