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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.

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes., The decision of whether and when a cell divides is tightly controlled. Here, the authors show in yeast that there is a multi-step competition between different phosphorylation states and sites in the S phase CDK-Sic1 complex, which controls Sic1 degradation and coordinates the precise timing of the G1/S transition.

The emergence of biological phosphate esters of glycerol could have been a crucial step in the origin and evolution of life on the early Earth as glycerol phosphates today play a central role in biochemistry. We investigate here the formation of the glycerol phosphates by employing various rock samples, salts, and minerals as potential catalysts to aid the phosphorylation process. We report the synthesis of various phosphate esters of glycerol including glycerol-1-phosphate, glycerol-2-phosphate, cyclic glycerol-monophosphate as well as various diphosphate esters. Furthermore, the decomposition rates of glycerol phosphate under mild heating were also studied while keeping the pH constant. It was observed that glycerol phosphate starts decomposing quickly under mild heating conditions into inorganic orthophosphate and pyrophosphate, and a steady state concentration of ~0.5 M of glycerol phosphate may have been reasonable in ponds with abundant glycerol, phosphate, urea, and catalytic minerals.

Protein kinases modulate the structure and function of proteins by adding phosphate groups to threonine, tyrosine, and serine residues. The phosphorylation process mediated by the kinases regulates several physiological processes, while their overexpression results in the development of chronic diseases, including cancer. Targeting of receptor tyrosine kinase pathways results in the inhibition of angiogenesis and cell proliferation that validates kinases as a key target in the management of aggressive cancers. As such, the identification of protein kinase inhibitors revolutionized the contemporary anticancer therapy by inducing a paradigm shift in the management of disease pathogenesis. Contemporary drug design programs focus on a broad range of kinase targets for the development of novel pharmacophores to manage the overexpression of kinases and their pathophysiology in cancer pathogenesis. In this review, we present the emerging trends in the development of rationally designed molecular inhibitors of kinases over the last five years (2016-2021) and their incipient role in the development of impending anticancer pharmaceuticals.

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.

International audience; Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

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.

International audience; Relative concentrations of alpha(S1)-casein and alpha(S2)-casein (alpha(S1)-CN and alpha(S2)-CN) phosphorylation isoforms vary considerably among milk of individual cows. We estimated heritabilities for alpha(S2)-CN phosphorylation isoforms, determined by capillary zone electrophoresis from 1,857 morning milk samples, and genetic correlations among alpha(S2)-CN phosphorylation isoforms in Dutch Holstein Friesian. To investigate if phosphorylation of alpha(S1)-CN and alpha(S2)-CN are due to the same genetic mechanism, we also estimated genetic correlations between alpha(S1)-CN and alpha(S2)-CN phosphorylation isoforms as well as the genetic correlations between the phosphorylation degrees (PD) of alpha(S1)-CN and alpha(S2)-CN defined as the proportion of isoforms with higher degrees of phosphorylation in total alpha(S1)-CN and alpha(S2)-CN, respectively. The intra-herd heritabilities for the relative concentrations of alpha(S2)-CN phosphorylation isoforms were high and ranged from 0.54 for alpha(S2)-CN-10P to 0.89 for alpha(S2)-CN-12P. Furthermore, the high intra-herd heritabilities of alpha(S1)-CN PD and alpha(S2)-CN PD imply a strong genetic control of the phosphorylation process, which is independent of casein production. The genetic correlations between alpha(S2)-CN phosphorylation isoforms are positive and moderate to high (0.33-0.90). Furthermore, the strong positive genetic correlation (0.94) between alpha(S1)-CN PD and alpha(S2)-CN PD suggests that the phosphorylation processes of alpha(S1)-CN and alpha(S2)-CN are related. This study shows the possibility of breeding for specific alpha(S1)-CN and alpha(S2)-CN phosphorylation isoforms, and relations between the phosphorylation degrees of alpha(S1)-CN and alpha(S2)-CN and technological properties of milk need to be further investigated to identify potential benefits for the dairy industry.

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.

Deoxyadenosine kinase from Dictyostelium discoideum (DddAK) phosphorylates its natural substrate (2’-deoxyadenosine, dAdo) as well as the arabinosyladenine analogues vidarabine (araA) and fludarabine (F-araA) to their corresponding 5’-monophosphates. DddAK has been here immobilized by ionic interaction on an aminated epoxy-functionalized support (SepabeadsTM EC-EP), and cross-linked with oxidized dextran. The final activity recovery was 33–42 %, depending on the protein loading. Immobilization enhanced the stability of DddAK at pH 10 and, to a lesser extent, at 45 °C. Phosphorylation of dAdo, araA and F-araA catalyzed by immobilized DddAK was always nearly quantitative in less than 12 hours. Fludarabine monophosphate was synthesized from F-araA (95 % conversion, 20 g/L) using immobilized DddAK in fully aqueous medium, thus showing that this biocatalyst could be used for developing a preparative phosphorylation process greener and more efficient than POCl3-based phosphorylation. (Less)

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.

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. Fil: Alvau, Antonio. University of Massachussets; Estados Unidos Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gervasi, Maria Gracia. University of Massachussets; Estados Unidos Fil: Navarrete, Felipe A.. University of Massachussets; Estados Unidos Fil: Xu, Xinran. State University of Colorado - Fort Collins; Estados Unidos Fil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: De la Vega Beltran, José Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Greer, Peter. Queens University; Canadá Fil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Krapf, Diego. State University of Colorado - Fort Collins; Estados Unidos Fil: Salicioni, Ana María. University of Massachussets; Estados Unidos Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Visconti, Pablo E.. University of Massachussets; Estados Unidos

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.

A novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticles as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after the phosphorylation process in the presence of adenosine 59-[c-thio] triphosphate (ATP-s) cosubstrate. Due to its good conductivity, large surface area, and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of 0.07 U mL(-1), wide linear range (from 0.07 to 32 U mL(-1)), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC(50) of ellagic acid, a PKA inhibitor, was estimated, which was in agreement with those characterized with the conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor, but not PKA inhibitor, shows its excellent performance in kinase inhibitor screening. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.

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.

Transient exposure of immature animals during the brain growth spurt period to ethanol triggers neuroapoptosis in the developing brain. Here we report that lithium, when administered in a single, well-tolerated dose to infant mice, suppresses spontaneous neuroapoptosis that occurs naturally in the developing brain, and prevents ethanol from triggering neuroapoptosis. To explore lithium's mechanism of action, we focused on kinase signaling systems (ERK, Akt, JNK) that are believed to play a regulatory role in cell survival, and found that very rapidly after ethanol administration there is a suppression of ERK phosphorylation, and that lithium stimulates ERK phosphorylation and prevents ethanol from suppressing this phosphorylation process. Ethanol also suppressed pAKT, but lithium did not counteract this effect. We also found that ethanol activates the JNK system, but this cannot explain the neurotoxic action of ethanol, because JNK activation did not occur in the same neuronal populations that are killed by ethanol.

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.

Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

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.