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By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.

The conserved three-dimensional structure of receptor tyrosine kinases (RTKs) has been varyingly observed in prokaryotes to humans that actively participate in the phosphorylation process of tyrosine residues in the protein, which results in the alteration of protein's function. Mutation and transcriptional or post-translational modifications lead to a deregulation of kinases, which ultimately fallout into the development of pathological conditions like cancer. The human genome encodes two kinds of tyrosine kinases: non-receptor tyrosine kinases (NRTKs) and receptor tyrosine kinases (RTKs). Among these kinases, VEGF/VEGFR-2 signaling cascade is an important target to develop novel small-molecule inhibitors for the therapy of abnormal angiogenesis incorporated with cancer. Due to advances in the knowledge of the catalytic domain and 'DFG-motif' region, selective 'DFG-in' (type I) and 'DFG-out' (type II) VEGFR-2/KDR inhibitors were successfully developed, and some are in different phases of a clinical trial. 'DFG-out' (inactive) confirmation has significant advantages over 'DFG-in' (active) confirmation concerning the affinity of the ATP at the catalytic domain. Further, in the catalytic domain, between front and back cleft, smaller gatekeeper residue (Val916) present; therefore, selectivity against VEGFR-2 could be precisely achieved. In this review, small molecule type II/'DFG-out' inhibitors, their conformation, interaction at receptor binding pocket, and structural requirements to inhibit VEGFR-2 at the molecular level are discussed.HighlightsVEGFR-2 is a type of membrane-bound receptor tyrosine kinases (RTKs) that regulates the process of vasculogenesis and angiogenesis.Small molecule first-generation type I, 'DFG-in' and second-generation type II, 'DFG-out' VEGFR-2 inhibitors exhibit clinical benefits in the treatment of aberrant angiogenesis associated with cancer.Molecular docking of FDA approved and novel type II inhibitors were performed using X-ray crystal structures of VEGFR-2; binding site analysis was carried out.Structural requirements for the inhibition of VEGFR-2 were identified.

Replication of the genotype 2 hepatitis C virus (HCV) requires hyper-phosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyper-phosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyper-phosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in the above sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wildtype NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction, too, abolished NS5A hyper-phosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyper-phosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant that resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyper-phosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated auto-cleavage at the NS3-4A junction is critical to NS5A hyper-phosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyper-phosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.Importance For twenty some years, the HCV protease NS3 was implicated in NS5A hyper-phosphorylation. We now show that it was the NS3-mediated cis-cleavage at the NS3-4A junction that permits NS5A hyper-phosphorylation at serine 2201, 2208, 2211, and 2214, leading to hyper-phosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at the above serine residues right after NS3-4A cleavage and before NS5A was released from the NS4A-5A polyprotein. Our data suggest that the dual functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyper-phosphorylation.

The low cost, environmental friendliness, and reproducibility of kraft lignin (KL) make it a potential candidate for the development of new green material. The phosphorylation of KL can extend its application as a flame-retardant material. Herein, the phosphorylated kraft lignin (PKL) was systematically fabricated in a sustainable process by utilizing a green phosphating reagent, NH4H2PO4, in the presence of urea. The influence of the reaction parameters, i.e., reaction time and temperature, and NH4H2PO4/lignin ratio on the phosphorylation process were investigated. Advanced characterization techniques including 1H NMR, 31P NMR, and XPS confirmed that the phosphorus groups were successfully introduced to lignin molecules. The active phenolic and aliphatic hydroxy groups of kraft lignin underwent a nucleophilic substitution reaction with the phosphate group to generate phosphorylated lignin. Compared with KL, PKL showed excellent thermal stability, and its maximum decomposition temperature was 620 °C compared with 541 °C for KL.

Legume seed development represents a high demand for energy and metabolic resources to support the massive synthesis of starch and proteins. However, embryo growth occurs in an environment with reduced O2 that forces the plant to adapt its metabolic activities to maximize efficient energy use. SNF1-related protein kinase1 (SnRK1) is a master metabolic regulator needed for cells adaptation to conditions that reduce energy availability, and its activity is needed for the successful development of seeds. In bean embryo extracts, SnRK1 can be separated by anion exchange chromatography into two pools: one where the catalytic subunit is phosphorylated (SnRK1-p) and another with reduced phosphorylation (SnRK1-np). The phosphorylation of the catalytic subunit produces a large increase in SnRK1 activity but has a minor effect in determining its sensitivity to metabolic inhibitors such as trehalose 6-P (T6P), ADP-glucose (ADPG), glucose 1-P (G1P) and glucose 6-P (G6P). In Arabidopsis thaliana, upstream activating kinases (SnAK) phosphorylate the SnRK1 catalytic subunit at T175/176, promoting and enhancing its activity. Recombinant Phaseolus vulgaris homologous to SnAK proteins (PvSnAK), can phosphorylate and activate the catalytic domains of the α-subunits of Arabidopsis, as well as the SnRK1-np pool purified from bean embryos. While the phosphorylation process is extremely efficient for catalytic domains, the phosphorylation of the SnRK1-np complex was less effective but produced a significant increase in activity. The presence of SnRK1-np could contribute to a quick response to unexpected adverse conditions. However, in addition to PvSnAK kinases, other factors might contribute to regulating the activation of SnRK1.

A simple strategy to prepare cost-effective adsorbent materials for the removal of U(VI) in radioactive wastewater is of great significance to environmental protection. Here, activated orange peel was used as a precursor for the synthesis of biomass charcoal, and then a phosphorylated honeycomb-like porous carbon (HLPC-PO4) material was prepared through simple phosphorylation modification. FT-IR and XPS showed that P–O–C, P–C, and P˭O bonds appeared in HLPC-PO4, indicating that the phosphorylation process is mainly the reaction of C–O bonds on the surface of the material with –PO4. The results of the batch experiments showed that the uptake equilibrium of HLPC-PO4 to U(VI) occurred within 20 min, and the kinetic simulation showed that the process was monolayer chemical adsorption. Interestingly, the maximum U(VI) uptake capacity of HLPC-PO4 at T = 298.15 K and pH = 6.0 was 552.6 mg/g, which was more than 3 times that of HLPC. In addition, HLPC-PO4 showed an adsorption selectivity of 70.1% for U(VI). After 5 cycles, HLPC-PO4 maintained its original adsorption capacity of 90.5%. The adsorption mechanism can be explained as the complexation of U(VI) with P–O and P˭O on the surface of the adsorbent, confirming the strong bonding ability of –PO4 to U(VI).

Background Annexin A2 (AnxA2) is a multifunctional protein involved in endocytosis, exocytosis, membrane domain organisation, actin remodelling, signal transduction, protein assembly, transcription and mRNA transport, as well as DNA replication and repair. Scope of review The current knowledge of the role of phosphorylation in the functional regulation of AnxA2 is reviewed. To provide a more comprehensive treatment of this topic, we also address in depth the phosphorylation process in general and discuss its possible conformational effects. Furthermore, we discuss the apparent limitations of the methods used to investigate phosphoproteins, as exemplified by the study of AnxA2. Major conclusions AnxA2 is subjected to complex regulation by post-translational modifications affecting its cellular functions, with Ser11, Ser25 and Tyr23 representing important phosphorylation sites. Thus, Ser phosphorylation of AnxA2 is involved in the recruitment and docking of secretory granules, the regulation of its association with S100A10, and sequestration of perinuclear, translationally inactive mRNP complexes. By contrast, Tyr phosphorylation of AnxA2 regulates its role in actin dynamics and increases its association with endosomal compartments. Modification of its three main phosphorylation sites is not sufficient to discriminate between its numerous functions. Thus, fine-tuning of AnxA2 function is mediated by the joint action of several post-translational modifications. General significance AnxA2 participates in malignant cell transformation, and its overexpression and/or phosphorylation is associated with cancer progression and metastasis. Thus, tight regulation of AnxA2 function is an integral aspect of cellular homeostasis. The presence of AnxA2 in cancer cell-derived exosomes, as well as the potential regulation of exosomal AnxA2 by phosphorylation or other PTMs, are topics of great interest.

Mitogen-activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three-tiered kinase module-MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase-that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine-glutamic acid-tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal-regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)-selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID-SRM-MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono-phosphorylatable mutants ERKT202A and ERKY204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.

Synthesizing highly efficient adsorbent materials to remove U(VI) from contaminated water remains a major challenge. Here, using UiO66-NH2 as a sacrificial template, porous carbons (ZrO2@PCs) were prepared by high-temperature calcination, and then a simple strategy was used for phosphorylation modification to prepare the target product ZrO2@PCs-PO4. The microscopic surface morphology and structure of ZrO2@PCs-PO4 characterized by SEM, FT-IR and XRD reveals that phosphate groups has been successful introduced on the surface of ZrO2@PCs-PO4. Batch experimentation showed that the phosphorylation process improves the adsorption behaviors, and the maximum monolayer adsorption capacity for U(VI) is 496.5 ​mg/g. The adsorption isotherms and kinetics were simulated to explore its adsorption mechanism. The excellent selectivity and reusability ensure this material could apply in selective removal of U(VI) ions.

In this study, porcine bone collagen peptide (CP) was phosphorylated with sodium tripoly phosphate (STP) in order to improve its calcium binding capacity and osteogenic activity. Firstly, the phosphorylation process were optimized by an orthogonal experiment. Then, the effects of CP, phosphorylated CP (PCP), CP-calcium chelate (CP-Ca) and PCP-Ca on proliferation, differentiation and mineralization of MC3T3-E1 cells were investigated. The results showed that all of the CP, PCP, CP-Ca and PCP-Ca could enhance the proliferation, differentiation and mineralization of MC3T3-E1 cells by increasing the mRNA expression levels of alkaline phosphatase (ALP), Runt-related transcription factor-2 (Runx2), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), as well as the protein expression levels of Runx2 and β-Catenin. Especially, PCP-Ca had the greatest and significant effect on mineralization of MC3T3-E1 cells (P

The collapsin response mediator protein (CRMP-2) is hyperphosphorylated in Alzheimer's disease (AD). These phosphorylation events are mediated by specific kinase proteins, GSK3β and Cdk5, and occur at target phosphorylation sites majorly located at the C-terminal tail of CRMP-2. The abilities of naringenin (NAR) and naringenin-7-O-glucuronide (NAR-7-O-G) to selectively bind CRMP-2 and reduce its phosphorylation have been previously demonstrated; the molecular interplay between these events remains unresolved. Using computational tools, we unravel the possible mechanisms by which these molecules disrupt CRMP-2 phosphorylation. Structural and dynamic analyses revealed that while the C-terminal tail of unbound CRMP-2 was extended and subtly organized, notable conformational disarray and rigidity characterized this region when bound by NAR and NAR-7-O-G. Consequentially, atomistic motions of constituent phosphorylation sites were restricted, indicative of structural occurrences that could distort the accessibility of interactive kinase proteins. A similar pattern was observed at a target phosphorylation site located in the globular domain of CRMP-2. MM/PBSA analyses revealed that both compounds interacted favorably with CRMP-2 while crucial residues that enhanced their selective binding include Glu353, Thr349, Lys254, Asp140 and Arg75. These structural insights provide mechanistic events that could contribute towards the structure-based design of anti-AD molecules which can bind CRMP2 selectively and alter its phosphorylation process.

Phosphorylation of eukaryotic initiation factors was previously shown to interact with m7G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m7G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m7-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22 °C. Phosphorylated and non-phosphorylated eIFiso4E-m7G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m7-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m7-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m7-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants.

Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150 kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the interplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.

Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders.

Sugars are not only important energy sources and structural components, but they also act as signaling molecules that are involved in specific signal sensing. Among all ketohexose analogs (d-fructose, d-psicose, d-tagatose, d-sorbose, l-fructose, l-psicose, l-tagatose and l-sorbose), only d-psicose inhibited the growth of Arabidopsis roots. Phosphorylation by fructokinase occurred in d-fructose and d-psicose. d-Psicose-induced inhibition was relieved by adding d-fructose. Thus, the inhibition could not be attributed to the toxicity of phosphorylated d-psicose. The phosphorylation process requires ATP. After phosphorylation, d-fructose is metabolized in glycolysis and becomes energy sources and structural components, whereas d-psicose cannot contribute to the energy sources and structural components because it does not get metabolized further. However, d-psicose did not affect ATP level in the Arabidopsis roots, suggesting that the d-psicose-induced growth inhibition may not be related to the starvation of ATP. The phosphorylation of ketohexoses by fructokinase is known to a trigger signal-sensing resulting in growth inhibition. Therefore, d-psicose can be phosphorylated by fructokinase and this process may play a possible role in signal-sensing. This is probably one of the useful model systems for the study of the hexokinase-independent sugar-sensing function and for developing new types of weed-control agents.

The one-pot preparation of azanucleotide analogues from proline derivatives has been achieved by a sequential radical decarboxylation/phosphorylation process. The process proceeded under mild conditions, giving high yields of the nucleotide analogues. Remarkably, the stereoselectivity of the reaction can be controlled by using different oxygen and nitrogen substituents at C-4 to give preferentially either the 2,4-cis or 2,4-trans products. In this way, the diastereomeric cis/trans ratio can be shifted from 98:2 to 15:85.

An efficient metal-free phosphorylation process based on a Sandmeyer-type transformation with arylamines as the starting materials is developed. The transformation proceeds smoothly at room temperature without the exclusion of moisture or air. This phosphorylation reaction tolerates a wide range of functional groups and affords the phosphorylation products in moderate to good yields, thus providing a valuable method for the formation of aromatic carbon-phosphorus bonds.

Background Phosphorylation in the cell nucleus is closely associated with cell death during the cell-division cycle. Since the discovery that the phosphorylation process is inhibited by radiation exposure, it has been believed that radiation exposure causes disorder in the electron transfer system. It is believed that vitamin C activates the electron transfer system. Vitamin C has been confirmed to kill cancer cells in vitro. However, the amount of vitamin C necessary to kill cancer cells in vivo is not known, because vitamin C is needed by normal cells, and it is difficult to determine its concentration since doing so requires determination of the circulating blood volume, extracellular fluid volume, intracellular fluid volume, osmotic pressure, etc. In addition, while thermotherapy has potential in combination with radiation therapy or chemotherapy, further quantitative analysis is necessary. Methods and results In thought experiments, if a fundamental particle collides with (or is scattered by) nucleons or photons, the electrical attraction produced by protons and electrons is 1.07 × 10 −15 kg. However, this value will vary, because it depends on the energy of light. There also remains the problem of the unit. Conclusion It is believed that the chemical shift can be used to integrate physical and chemical study through the study of cyclotrons and magnetic resonance imaging (MRI). The origin of this result is that the mass and charge of the photon are zero. This means that when composite particles are obtained or lost, the energy produced by collisions of the photons with a receiver can be taken as a signal. Adenosine triphosphate (ATP) can be used to embody this concept in a molecule. The relationship between time and the ATP concentration becomes increasingly important in considering this question. Furthermore, when ATP is considered in this way, a problem arises with energy accounting, as well as with the concentration of vitamin C. A significant proposal resulting from this concept is that chemical shifts can be used in clinical practice. It is believed that the magnitude of the ATP signal detected by electron microscopy can be combined with the electrical quantitative analysis obtained by magnetic resonance spectroscopy (MRS) when it is calculated as an approximation of the coupling constant of protons and electrons.

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.

Protein phosphorylation plays an important role in intracellular signaling. Defects in the phosphorylation process, which are associated with cancers and other human diseases, can be reflected by the presence of abnormally phosphorylated peptides in serum. Therefore, phosphorylated peptides are important resources for biomarker discovery. However, few studies have been reported on developing sensors for phosphorylated peptides. In this study, zinc(ii) dipicolylamine-appended gold nanoparticles were synthesized and used for the colorimetric detection of bis-phosphorylated peptides. The addition of the bis-phosphorylated peptides could induce the aggregation of the functionalized AuNPs, and thus the color change of the gold colloid, while non-phosphorylated and mono-phosphorylated controls could not induce the color change.

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.

An increase in the health and environment legislation awareness pushed the textile manufacturers to develop their strategies to produce eco-friendly flame-retardant textiles with competitive cost. Chitosan is added during the phosphorylation process as a nitrogen source that has synergistic effect with phosphorus. Increasing the chitosan concentration from 0% to 2% enhances the flame retardancy of the treated cotton fabric against successive washing. Further increase in amount of chitosan, above 2%, has a limited effect on the thermal degradation of the treated cotton. Increasing diammonium hydrogen phosphate (DAHP) concentration from 0% to 10% is responsible for making the flame-retardant finish durable against successive washing. Thermal degradation analysis of treated fabrics at different DAHP concentrations shows a decrease in the maximum degradation rate point and thermal degradation onset point. The amount of residue at 500°C increases till reaches 32% with the increase in the DAHP concentr...

Sodium stannate/phosphate is an ideal eco-friendly flame retardant agent for cotton fabric. Development of this technique is an essential way to overcome some of its disadvantages such as the harsh feeling as a result of using high concentration of sodium stannate. Chitosan is added in the phosphorylation bath as a nitrogen source and to facilitate the phosphorylation process. Incorporation of 1% chitosan could decrease the sodium stannate concentration to the one-third of the amount that is used in the conventional method. Increasing the stannate concentration in the finishing bath from 10 to 30% could enhance the flame retardancy of the cotton fabric. Thermogravimetric analysis of the treated cotton fabric shows an increase in the residual percent of the fabric and decrease in both thermal degradation onset point (TDOP) and maximum degradation rate point as a function of stannate concentration. Increasing diammonium hydrogen phosphate (DAHP) from 2 to 10% in the finishing bath shows an increase...

Within the histopathology of Alzheimer's disease (AD) certain hallmarks are beeing observed. The occurance of protein deposits belong to such characteristic features. Such deposits can be found extracellular as beta-amyloid (Abeta) plaques and intracellular as neurofibrillary tangles (NFTs). In the search for novel AD therapeutics it became of great interest to investigate the formation of NFTs and their contribution to the AD symptomatic. NFTs consist of hyperphosphorylated tau protein. Within the phosphorylation process of tau protein two kinases are of great importance: cyclin dependent kinase 5 (cdk5) and its truncated regulatory subunit p25 and glycogen synthase kinase 3beta (GSK-3beta). The role of both kinases within the NFT formation process is still under debate. To better understand the pathophysiological process highly selective inhibitors of both kinases are of value. Known inhibitors lack the necessary selectivity. We developed novel 1-aza-9-oxafluo-renes as selective GSK-3beta inhibitors. Structure-activity relationships of a series of 4-phenyl substituted derivatives are discussed. Variation of the 3-side chain led to selective carbonyl amide derivatives with selectivity factors of more than 100 at the tested ATP competitor concentrations. Such selectivities permit specific investigation of the role of GSK-3beta within the NFT formation processes.

Kinetics of the reaction of alkylated polyethylene imines with 4-nitrophenyl bis(chloromethyl)-phosphinate in chloroform in the absence and in the presence of substituted calix[4]resorcinarenes were studied by UV spectrophotometry. It was shown that calixarenes forming mixed aggregates with polyethylene imines have a catalytic effect on the polymer phosphorylation process. The catalytic effect depends on the structure of polyethylene imine and the component ratio in the system.

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system.

Regulation of NF-kappaB activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kappaB activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kappaB signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes with the IKK, NF-kappaB, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kappaB transcriptional activity. In contrast to IKK and NF-kappaB, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNFalpha stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kappaB pathway.

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C4 phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C4 plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO2 concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C4 PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP3 levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP3 and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.

This study investigated the regulation of major light harvesting chlorophyll a/b protein (LHCII) phosphorylation by hypoosmotic shock in dark-adapted Dunaliella salina cells. When the external NaCl concentration decreased in darkness, D. salina LHCII phosphorylation levels transiently dropped within 20 min and then restored gradually to basal levels. The transient decrease in LHCII phosphorylation levels was insensitive to NaF, a phosphatase inhibitor. Inhibition of intracellular ATP production by addition of an uncoupler or an ATP synthase inhibitor increased LHCII phosphorylation levels in D. salina cells exposed to hypoosmotic shock. Taken together, these results indicate that hypoosmotic shock inhibits the LHCII phosphorylation process. The related mechanism and physiological significance are discussed.

It has been reported that gonadotropins promoted phosphorylation of ERK/MAPK in granulosa cells. However, little is known about the effects of gonadotropin on ERK activity in theca cells. This study explores how LH/forskolin controls ERK phosphorylation in cultured bovine theca cells. Effects of ERK on steroidogenesis were also investigated. Phosphorylation of ERK in bovine theca cells was augmented by LH and forskolin in 5 min; it decreased thereafter below basal levels in 20 min. Nevertheless, phosphorylation of the ERK kinase, MEK, was unaffected. Addition of H89 (a protein kinase A inhibitor) significantly reduced the effect of LH/forskolin on ERK phosphorylation. A potent MEK inhibitor PD98059 eliminated ERK phosphorylation and augmented progesterone production concomitantly with the elevation of intracellular steroidogenic acute regulatory protein mRNA in LH/forskolin-stimulated theca cells. In contrast to progesterone production, androgen production was diminished significantly by inhibition of ERK with decreased intracellular P450c17 mRNA levels. Taking these results together, we conclude that LH/cAMP leads to phosphorylation of ERK in a biphasic manner through MEK-independent pathway in bovine theca cells. Protein kinase A-induced phosphatase could possibly contribute to the phosphorylation process. Furthermore, modulation of ERK phosphorylation involves control of thecal steroidogenesis via modulation of the expression of steroidogenic acute regulatory protein and P450c17.

Eukaryotic cells use signal-transduction network to respond in specific ways to external signals from their environment. Several signal-transduction pathway are composed of multi-step chemical reactions. We here theoretically study what determines the number of kinase phosphorylation steps composing of the intracellular signal-transduction cascade. We examine a simple mathematical model for the association and phosphorylation process of kinases in the signal-transduction cascade. We focus on the speed of signal transduction as the criterion for determining the optimal response. The present model first reveals that the initial expression level of kinase in each step of the cascade must be the same in the optimal response under the constraint of the constant total kinase concentration. The second conclusion is that the optimal step number of kinase cascade is primarily determined by the ratio of the target concentration of the final phosphorylated kinase in the cascade to that of input signal molecule, C/S. A multi-step cascade is optimum if the amplification of the final product concentration C relative to the input signal S is sufficiently large; whereas, a single-step reaction is optimum if C/S is small. This suggests that multi-step phosphorylation would have evolved to accelerate the speed of transduction of weak signals.

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

Cation-exchange systems were prepared by phosphorylation of some lignocellulosic materials, namely, rice straw, cotton stalks, and bagasse. The effect of the particle size of the lignocellulosic materials as well as their chemical constituents on the phosphorylation process was studied by the determination of the phosphorus content of the prepared ion-exchange material. Phosphorus was determined using inductively coupled plasma atomic emission spectrometry. Optimization of the phosphorylation reaction was achieved by studying the effect of the different experimental parameters, namely, the reaction time, temperature, and amount of phosphorus oxychloride added, on the phosphate content of the reaction product. The treatment of the lignocellulosic material with sodium hydroxide was found to improve its phosphorylation. The cation-exchange efficiency of the produced phosphated material toward Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Ni2+, Pb2+, and Zn2+ was examined. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 792–800, 2002

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a / b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.

Microinjection of the restriction endonuclease HaeIII, which causes DNA double-strand breaks with blunt ends, induces nuclear accumulation of p53 protein in normal and xeroderma pigmentosum (XP) primary fibroblasts. In contrast, this induction of p53 accumulation is not observed in ataxia telangiectasia (AT) fibroblasts. HaeIII-induced p53 protein in normal fibroblasts is phosphorylated at serine 15, as determined by immunostaining with an antibody specific for phosphorylated serine 15 of p53. This phosphorylation correlates well with p53 accumulation. Treatment with lactacystin (an inhibitor of the proteasome) or heat shock leads to similar levels of p53 accumulation in normal and AT fibroblasts, but the p53 protein lacks a phosphorylated serine 15. Following microinjection of HaeIII into lactacystin-treated normal fibroblasts, lactacystin-induced p53 protein is phosphorylated at serine 15 and stabilized even in the presence of cycloheximide. However, neither stabilization nor phosphorylation at serine 15 is observed in AT fibroblasts under the same conditions. These results indicate the significance of serine 15 phosphorylation for p53 stabilization after DNA double-strand breaks and an absolute requirement for ATM in this phosphorylation process.

Amidotriphosphate (0.1 M) in aqueous solution at near neutral pH in the presence of magnesium ions (0.25 M) converts glycolaldehyde (0.025 M) within days at room temperature into glycolaldehyde phosphate in (analytically) nearly quantitative yields (76% in isolated product). This robust phosphorylation process was observed to proceed at concentrations as low as 30 microM glycolaldehyde and 60 microM phosphorylation reagent under otherwise identical conditions. In sharp contrast, attempts to achieve a phosphorylation of glycolaldehyde with cyclotriphosphate ('trimetaphosphate') as phosphorylating reagent were unsuccessful. Mechanistically, the phosphorylation of glycolaldehyde with amidotriphosphate is an example of intramolecular delivery of the phosphate group.

The role of phosphorylation in modulating an inwardly rectifying ATP-regulated K+ channel with inward conductance of about 90 pS was examined using the patch-clamp technique on opossum kidney (OK) cells. The activity of the inwardly rectifying K+ channel observed in cell-attached patches rapidly declined (channel "rundown") upon excision of the membrane into inside-out patches in a control bath solution (3 mM Mg2+, ATP-free). The declined channel activity was partially restored by applying ATP to the bath, and the ATP-induced channel restoration reached the near maximal level at an ATP concentration of 3 mM. The channel activity maintained by 3 mM ATP in inside-out patches was inhibited by K-252a (10 microM), a nonspecific protein kinase inhibitor, or KT5720 (200 nm), a specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), and was further stimulated by the addition of a catalytic subunit of PKA (20 nM). In cell-attached patches, the channel activity was also inhibited by K-252a (10 microM) or KT5720 (200 nM). The application of dibutyryl-cAMP (100 microM) alone failed to enhance channel activity, but significantly stimulated channel activity after the pretreatment of cells with Ro-20-1724 (100 microM), an inhibitor of cAMP-specific phosphodiesterase. These results suggest that maintenance of the activity of ATP-regulated K+ channels in OK cells requires protein kinase-mediated phosphorylation with ATP-hydrolysis, and that phosphorylation is mainly induced by PKA.

Photosystem II (PSII) is a primary target for light-induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo-oxidative damage. We propose that PSII core phosphorylation contributes to fine-tuned degradation of D1.

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [γ-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

In contrast to both its parent “troika” acid ( E - 1 , a phosphorylating agent at pH 7 and 25 °C) and its C-methyl isomer ( E - 2 , which is stable at both acidic and neutral pH), ( E )-(hydroxyimino)(hydroxymethoxyphosphinyl)acetic acid E - 3 was unreactive at pH 7 and 25 °C but at pH 1.5 fragmented to methyl phosphate 10 (15%) and methyl phosphorocyanidate 11 (85%). The minor product is consistent with solvent phosphorylalion, the reaction exclusively observed with E - 1 . The non-phosphorylating fragmentation pathway is proposed to involve a preliminary E → Z isomerizalion of 3 prior to C α -C β cleavage. Dual fragmentation pathways were also detected ( 31 P NMR) when the DCHA + salt of E - 3 ( E - 9 ) was heated in acetonitrile or EtOH; in addition to phosphorylation products (16–19%), 11 was formed (81–84%). Reaction of E - 9 in refluxing EtOH: t -BuOH (1:1) showed low stereoselectivity in product formation (~3:1 ethyl methyl phosphate: t -butyl methyl phosphate), supporting a dissociative phosphorylation process.

This study has demonstrated for the first time the use of peptide inhibitor-protein kinase recognition in QCM technology for detecting protein kinase A (PKA). Because of its high affinity with the catalytic subunit of PKA, protein kinase inhibitor (PKI) peptide presented a novel and potent recognition element to detect active PKA. This PKI-based method is rapid (less than 10 min), facile, and reagentless due to its one-step detection without phosphorylation process, which exhibits unique advantages over conventional kinase assays that are time-consuming (normally more than 1 h), multi-steps, and requiring addition of several reagents in phosphorylation and detection process. The piezoelectric kinase sensor is capable of sensitively detecting protein kinase A with a detection limit of 0.061 mU μL−1 and monitoring the binding of PKA and PKI in real-time.

International audience; Phosphorylation of the microtubule-associated Tau protein plays a major role in the regulation of its activity of tubulin polymerization and/or stabilization of microtubule assembly. A dysregulation of the phosphorylation/dephosphorylation balance leading to the hyperphosphorylation of Tau proteins in neurons is thought to favor their aggregation into insoluble filaments. This in turn might underlie neuronal death as encountered in many neurodegenerative disorders, including Alzheimer's disease. Another post-translational modification, the O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), controls the phosphorylation state of Tau, although the precise mechanism is not known. Moreover, analytical difficulties have hampered the precise localization of the O-GlcNAc sites on Tau, except for the S400 site that was very recently identified on the basis of ETD-FT-MS. Here, we identify three O-GlcNAc sites by screening a library of small peptides sampling the proline-rich, the microtubule-associated repeats and the carboxy-terminal domains of Tau as potential substrates for the O-β-N-acetylglucosaminyltransferase (OGT). The in vitro activity of the nucleocytoplasmic OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Using phosphorylated peptides, we establish the relationship between phosphate and O-GlcNAc incorporation at these sites. Phosphorylation of neighboring residues S396 and S404 was found to decrease significantly S400 O-GlcNAcylation. Reciprocally, S400 O-GlcNAcylation reduces S404 phosphorylation by the CDK2/cyclinA3 kinase and interrupts the GSK3β-mediated sequential phosphorylation process.

Interleukin-8 (IL-8) such as LUCT (lung giant cell carcinoma-derived chemotactic protein), NAP (neutrophil activating protein) and MDNCF (monocyte- derived neutrophil chemotactic factor), and formylmethionyl- leucyl-phenylalanine (fMLP) are well-known chemoattractants for human polymorphonuclear leukocytes (PMNs) and are able to stimulate phosphorylation of 64-kd protein (p64) in these leukocytes. To elucidate the molecular mechanism of PMN activation with chemoattractants, we investigated the phosphorylation process of p64 in an intact cell. 32P-Labeled PMNs were stimulated with LUCT/IL-8, fMLP, leukotriene B4, or C5a, and phosphorylated proteins were analyzed by two-dimensional electrophoresis and autoradiography. A marked phosphorylation of p64 was observed after stimulation. A new spot of phosphorylated p64 (pp64) could be detected on the gel stained with Coomassie Brilliant Blue, indicating that the isoelectric point (pI) of p64 shifted from 5.3 to a more acidic pI by the phosphorylation forming pp64. The spot of pp64 was shown to be dephosphorylated to p64 by treatment with calf intestine alkaline phosphatase. Other proteins having molecular masses 82, 66, 58, 55 and 50 kd were also phosphorylated. The fMLP- stimulated phosphorylation was time-dependent and saturated within 5 min. Maximum stimulation was achieved with 10 nM fMLP. Phosphoamino acid analysis revealed phosphorylation of serine residues in pp64. Staurosporine (100 nM) and W-7 (100 μΜ) significantly inhibited the phosphorylation of p64, but H-7 slightly inhibited it. H-8 and herbimycin A did not effect phosphorylation. Phorbol myristate acetate was found to stimulate significantly. Protein kinase C did not stimulate the phosphorylation. These data suggest that protein kinase C and calmodulin-like protein are indirectly involved in the phosphorylation of p64 during chemoattractant-activation of PMN.

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5°C, the association and dissociation rate constants of Mg · ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 μM calcium and 5 mM MgCl2 (3–4x 106 M−1· s−1 and 9 s−1 respectively). Therefore, we have determined the true affinity for Mg · ATP (Kd= 3 μM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg · ATP binding is the rate limiting step in the phosphorylation process, and Mg · ATP dissociation is slower than dephosphorylation. The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two-step mechanism, both steps being accelerated by Mg · ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca2+ -ATPase, we showed that rate constants for calcium binding are always lower than those of Mg · ATP binding to an EGTA-incubated enzyme. We measured dissociation and association rate constants of Mg · ATP binding in the absence of calcium (k-1= 25 s−1 and k1= 7.5 106 M−1· s−1). This gives a Kd similar to that obtained by equilibrium measurements (3–4 μM). Both non-phosphorylated conformations of the enzyme have similar affinity for Mg · ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non-phophorylated enzyme for Mg · ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca2+-ATPase cycle, in which ATP is sequentially substrate and activator on a multiple-function single site.

In contrast to both its parent “troika” acid ( E - 1 , a phosphorylating agent at pH 7 and 25 °C) and its C-methyl isomer ( E - 2 , which is stable at both acidic and neutral pH), ( E )-(hydroxyimino)(hydroxymethoxyphosphinyl)acetic acid E - 3 was unreactive at pH 7 and 25 °C but at pH 1.5 fragmented to methyl phosphate 10 (15%) and methyl phosphorocyanidate 11 (85%). The minor product is consistent with solvent phosphorylalion, the reaction exclusively observed with E - 1 . The non-phosphorylating fragmentation pathway is proposed to involve a preliminary E → Z isomerizalion of 3 prior to C α -C β cleavage. Dual fragmentation pathways were also detected ( 31 P NMR) when the DCHA + salt of E - 3 ( E - 9 ) was heated in acetonitrile or EtOH; in addition to phosphorylation products (16–19%), 11 was formed (81–84%). Reaction of E - 9 in refluxing EtOH: t -BuOH (1:1) showed low stereoselectivity in product formation (~3:1 ethyl methyl phosphate: t -butyl methyl phosphate), supporting a dissociative phosphorylation process.

The Bcl-2-associated death promoter (BAD) protein, like many other BH3-only proteins, is known to promote apoptosis through the intrinsic mitochondrial pathway. Unlike the BH3-interacting domain death agonist (BID) protein, BAD cannot directly trigger apoptosis but, instead, lowers the threshold at which apoptosis is induced. In many mathematical models of apoptosis, BAD is neglected or abstracted. The work presented here considers the incorporation of BAD and its various modifications in a model of the tBID-induction of BAK (Bcl-2 homologous antagonist killer) or the tBID-induction of BAX (Bcl-2-associated X protein). Steady state equations are used to develop an explicit formula describing the total concentration level of tBID, guaranteed to trigger apoptosis, as a bilinear function of the total BAD concentration level and the total anti-apoptotic protein concentration level (usually Bcl-2 or Bcl-xL). In particular, the formula explains how the pro-apoptotic protein BAD lowers the threshold at which tBID induces BAK/BAX activation-reducing the level of total Bcl-2/Bcl-xL available to inhibit tBID signaling in the mitochondria. Attention is then turned to the experimental data surrounding BAD phosphorylation, a process known to inhibit the pro-apoptotic effects of BAD. To address this data, the phosphorylation process is modeled following two separate kinetics in which either free unbound BAD is the assumed substrate or Bcl-xL/Bcl-2-bound BAD is the assumed substrate. Bifurcation analysis and further analysis of the bilinear equation validate experiments, which suggest that BAD phosphorylation prevents irreversible BAK/BAX-mediated apoptosis, even when phosphorylation-induced dissociation of Bcl-xL/Bcl-2-bound BAD is blocked. It is also shown that a cooperative, even synergistic, removal of mitochondrial BAD is seen when both types of phosphorylation are assumed possible. The presented work, however, reveals that the balance between BAD phosphorylation and dephosphorylation modulates the degree to which BAD influences the signaling from tBID to BAK/BAX. Our model shows that both the mode(s) of phosphorylation and the BAD dephosphorylation rate become important factors in determining whether BAD influences the activation of the BAK/BAX signal or not. Such potential variations in the pro-apoptotic effects of BAD are used to explain some of the inconsistent experimental data surrounding BAD phosphorylation. Nonetheless, our model serves to evaluate BAD and its sensitizing effects on the tBID-induction of BAK/BAX and thus aid in predicting when the incorporation of BAD in an apoptosis signaling model is important and when it is not.

The effect of different substances on the phosphorylation of axonal membrane proteins was tested. The results obtained show that: 1. 1. Maximum activity is recorded around pH 7.8. 2. 2. The net phosphorylation state of proteins depends on the K + and Na + concentration of the reaction medium. It is optimized at low concentration of Ca ++ ions (10 −4 M) and very much enhanced by MnCl 2 (10 mM). No phosphorylation is detected in the absence of Mg ++ ions. 3. 3. Neurotropic compounds, such as tetrodotoxin, cocaine, meprylcaine, novocaine, novesine and tetracaine (10 −4 M), generally inhibit the phosphorylation process. The effect of some of these substances is modulated by the calcium ions. 4. 4. The phosphorylation of proteins is inhibited by cyclic AMP (5 × 10 −5 M) but is independent of cyclic GMP (5 × 10 −5 M) and calmodulin (50 μg/mg protein). 5. 5. Vinblastine (3 × 10 −5 M), vincristine (5 × 10 −5 M), colchicin (5 × 10 −5 M), cytochalasin B (10 −4 M) and ouabain (10 −3 M) do not significantly modify the phosphorylation level of proteins. The data are discussed in relation to the according to which the configuration of the protein, forming the site controlling the Na + ion flow during the nerve activity, depends on its phosphorylation level.

The impedance variation cycle (IVC) of axonal membranes has been described in terms of a dephosphorylation-phosphorylation cycle. It has been experimentally approached by preparing membranes from the walking nerves of crabs and from the main electric organ of Electrophorus electricus . Membrane proteins were solubilized in 1% Lubrol-PX and purified by chromatographic techniques. We have obtained a protein fraction which was phosphorylated by low [γ-32P]-ATP concentrations, ranging from 5 × 10−8M to 10−7M. This fraction of high molecular weight proteins has been partly characterized in denaturing conditions revealing the presence of several protein species with different specific phosphorylation activity. At this experimental stage the two types of investigated materials displayed distinct patterns of phosphorylation versus molecular weight. The influence of tetrodotoxin and veratridine, on the phosphorylation process is a rather complex inhibitory effect, now under further investigation.