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Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively., (© 2021 Federation of European Biochemical Societies.)

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus , these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro , and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks., (Copyright © 2021 American Society for Microbiology.)

The determination of signaling pathways and transcriptional networks that control various biological processes is a major challenge from both basic science and translational medicine perspectives. Because such analysis can point to critical disease driver nodes to target for therapeutic purposes, we combined data from phenotypic screening experiments and gene expression studies of mouse neurons to determine information flow through a molecular interaction network using a network propagation approach. We hypothesized that differences in information flow between control and injured conditions prioritize relevant driver nodes that cause this state change. Identifying paths likely taken from potential source nodes to a set of transcription factors (TFs), called sinks, we found that kinases are enriched among source genes sending significantly different amounts of information to TFs in an axonal injury model. Additionally, TFs found to be differentially active during axon growth were enriched in the set of sink genes that received significantly altered amounts of information from source genes. Notably, such enrichment levels hold even when restricting the set of source genes to only those kinases observed to support or hamper neurite growth. That way, we found a set of 71 source genes that send significantly different levels of information to axon growth-relevant TFs. We analyzed their information flow changes in response to axonal injury and their influences on TFs predicted to facilitate or antagonize axon growth. Finally, we drew a network diagram of the interactions and changes in information flow between these source genes and their axon growth-relevant sink TFs.

The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.

Protein kinase signaling networks stringently regulate cellular processes, such as proliferation, motility, and cell survival. These networks are also central to the evolution and progression of cancer. Accordingly, genetically encoded fluorescent biosensors capable of directly illuminating the spatiotemporal dynamics of kinase signaling in live cells are being increasingly used to investigate kinase signaling in cancer cells and tumor tissue sections. These biosensors enable visualization of biological processes and events directly in situ , preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Herein, we first review common design strategies for kinase activity biosensors, including signaling targets, biosensor components, and fluorescent proteins involved. Subsequently, we discuss applications of biosensors to study the biology and management of cancer. These versatile molecular tools have been deployed to study oncogenic kinase signaling in living cells and image kinase activities in tumors or to decipher the mechanisms of anticancer drugs. We anticipate that the diversity and precision of genetically encoded biosensors will expand their use to further unravel the dysregulation of kinase signaling in cancer and the modes of actions of cancer-targeting drugs., (© 2019 Lin et al.)

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg 2+ -ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg 2+ concentration for optimum activity (20-50 mm), the apparent binding of levoglucosan (K m =180 mm) and ATP (K m =1.0 mm), as well as the inhibition by ADP (K i =0.45 mm) and d-glucose-6-phosphate (IC 50 =56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4 C 1 → 1 C 4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H 2 production. As H 2 production is tightly linked to cell growth in T. kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. kodakarensis., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate., (Copyright © 2017. Published by Elsevier B.V.)

The purpose of this study was to detect Streptococcus mutans by using monoclonal antibodies (mAbs) against S. mutans that cause dental caries and compare the levels of the bacterium between the saliva of adolescents undergoing orthodontic treatment (OT) and those not undergoing treatment (NT). Saliva samples, collected from 25 OT adolescents (with a mean age of 12.84 years) and 25 NT adolescents (mean age of 12.4 years), were analyzed by Dentocult-SM and enzyme-linked immunosorbent assay using mAbs against Ag I/II (ckAg I/II) and GTF B (ckGTF B), GTF C (ckGTF C), and GTF D (ckGTF D) of S. mutans. The DMFT index was slightly higher in the OT group (5.12 in OT and 4.96 in NT) and the level of S. mutans (≥10 5 CFU/mL) was higher in OT (72%) than in NT (56%). The detected levels of ckAg I/II, ckGTF B, ckGTF C, and ckGTF D were slightly higher in OT than in NT. The results of this study indicate that use of mAbs against S. mutans yields sensitive detection for the bacterium in saliva samples and shows that it has a reliable connection to the number of S. mutans and decayed, missing, filled teeth (DMFT), suggesting that the levels of S. mutans in saliva can be defined and compared by the application of the mAbs.

P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases., From the Clinical Editor: This team of authors have utilized a self-assembled cell microarray to screen genes that regulate cancer cell migration using a human genome siRNA library of kinases. They validated previously known genes and identified novel ones that may serve as therapeutic targets., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

Key Message: We have expressed, purified, and biophysically characterized recombinant AHP1 and AHP2. Also, using computational homology models for AHP1, ARR7, and AHP1–ARR7 complex, we identified threedimensional positioning of key amino acids. Cytokinin signaling involves activation of Arabidopsis Response Regulators (ARRs) by Arabidopsis Histidine Phosphotransfer Proteins (AHPs) by phosphorylation. Type-A ARRs are key regulators of several developmental pathways, but the mechanism underlying this phosphorylation and activation is not known in plants. In this study, we report the successful expression and purification of recombinant AHP1 and AHP2. Biophysical characterization shows that these two recombinant proteins were purified to homogeneity and possess well-defined secondary structures. Brief attempts to purify recombinant ARR7 posed problems during size-exclusion chromatography. Nevertheless, we generated computational homology models for AHP1, ARR7, and AHP1-ARR7 complex using crystal structures of homologous proteins from other organisms. The homology models helped to identify the three-dimensional positioning of the key conserved residues of AHP1 and ARR7 involved in phosphorylation. The similarity in positioning of these residues to other homologous proteins suggests that AHPs and type-A ARRs could be structurally conserved across kingdoms. Thus, our homology models can serve as valuable tools to gain structural insights into the phosphorylation and activation of cytokinin response regulators in plants.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the β3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ∼1500 s(-1)) between major open (∼97%) and minor closed (∼3%) conformations. Upon PEP binding, the β3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.

The N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the k(ex) of ~500 s(-1) which compares to the phosphoryl transfer rate of ~850 s(-1) between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²⁺ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²⁺ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k (cat) and K(m) values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t(1/2) = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.

Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the ∼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Tuberculosis is a widespread and deadly infectious disease, with one third of the human population already being infected. Aminoglycoside antibiotics have become less effective in recent years owing to antibiotic resistance, which arises primarily through enzymatic modification of the antibiotics. The gene product Rv3168, a putative aminoglycoside phosphotransferase (APH), from Mycobacterium tuberculosis was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M calcium acetate, 0.1 M Tris-HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.67 Å on a synchrotron beamline. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.74, b = 62.37, c = 103.61 Å. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.91 Å(3) Da(-1). The structure was solved by the single-wavelength anomalous dispersion method and refinement of the selenomethionine structure is in progress.

Background: p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca(2+)-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood., Methodology/principal Findings: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca(2+) wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine., Conclusion: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.

An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver. In the present study, we combined the functional isolation of kinases with different separation workflows of automated split-free nanoflow liquid chromatography prior to mass spectrometric analysis. Two different CCMS setups, CCMS technology combined with 1D LC-MS and 2D LC-MS, were compared regarding the total number of kinase identifications. By extending the chromatographic separation of the tryptic digested captured proteins from 1D LC linear gradients to 2D LC we were able to identify 97 kinases. This result is similar to the 1D LC setup we previously reported but this time 4 times less input material was needed. This makes CCMS of kinases an even more powerful tool for the proteomic profiling of this important protein family.

The Embden-Meyerhof-Parnas (EMP) glycolysis is the starting point of the core carbon metabolism. Aerobic methanotrophs possessing activity of the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the classical glycolytic enzyme ATP-dependent 6-phosphofructokinase (ATP-PFK) are promising model bacteria for elucidation of the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in microorganisms. Characterization of the His(6)-tagged PPi-PFKs from two methanotrophs, halotolerant alkaliphilic Methylomicrobium alcaliphilum 20Z and thermotolerant Methylococcus capsulatus Bath, showed differential capabilities of PPi-PFKs to phosphorylate sedoheptulose-7-phosphate and this property correlated well with the metabolic patterns of these bacteria assimilating C(1) substrate either via the ribulosemonophosphate (RuMP) pathway (Mm. alcaliphilum 20Z) or simultaneously via the RuMP and serine pathways and the Calvin cycle (Mc. capsulatus Bath). Analysis of the genomic draft of Mm. alcaliphilum 20Z (https://www.genoscope.cns.fr/agc/mage) has provided in silico evidence for the existence of a PPi-dependent pyruvate-phosphate dikinase (PPDK). Expression of the ppdk gene at oxygen limitation along with the presence of PPi-PFK in Mm. alcaliphilum 20Z implied functioning of PPi-dependent glycolysis and PPi recycling under conditions when oxidative phosphorylation is hampered., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Quantitative mass spectrometry-based proteomic assays often suffer from a lack of robustness and reproducibility. We here describe a targeted mass spectrometric data acquisition strategy for affinity enriched subproteomes-in our case the kinome-that enables a substantially improved reproducibility of detection, and improved quantification via isobaric tags. Inclusion mass lists containing m/z, charge state, and retention time were created based on a set of 80 shotgun-type experiments performed under identical experimental conditions. For each target protein, peptides were selected according to their frequency of observation and isobaric tag for relative and absolute quantitation (iTRAQ) reporter ion quality. Retention times of selected peptides were aligned using similarity driven pairwise alignment strategy yielding <1 min standard deviation for 4 h gradients. Multiple fragmentation of the same peptides resulted in better statistics and more precise reporter ion based quantification without any loss in coverage. Overall, 24% more target proteins were quantified using the targeted data acquisition approach, and precision of quantification improved by >1.5-fold. We also show that a combination of higher energy collisional dissociation (HCD) with collisional induced dissociation (CID) outperformed pulsed-Q-dissociation (PQD) on the OrbitrapXL. With the CID/HCD based targeted data acquisition approach 10% more quantifiable target proteins were identified and a 2-fold increase in quantification precision was achieved. We have observed excellent reproducibility between different instruments, underlining the robustness of the approach., (Copyright © 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.)

The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show approximately 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.

NS5A phosphorylation can be studied in two ways: in living cells and in vitro. The former has several advantages: NS5A phosphorylation takes place in a cellular background and therefore might mimic more closely the real in vivo situation. Viral proteins and cellular kinases are in the correct cellular compartments, and dynamic processes like viral polyprotein processing and cellular signaling are in place. The disadvantage of this system is its great complexity, which makes limiting an observed effect to a single, well-defined agent, for example, a kinase, very difficult. NS5A phosphorylation in cells can easily be followed by metabolic labeling with either (35)S-methionine or (32)P-orthophosphate. The effect of a single, well-defined kinase on NS5A phosphorylation can be investigated in cells either by overexpression of this kinase in the presence of NS5A or by RNA interference of this kinase. If available, specific kinase inhibitors can be used to reveal the effect of this inhibition on NS5A phosphorylation. The problem with this approach is that only very few really specific kinase inhibitors are available. Biochemical in vitro experiments use purified components. This type of experiment allows direct investigation of the activity of a single kinase on NS5A as a substrate. In addition, the precise phosphorylation sites of a kinase can be mapped when NS5A-derived peptides are used instead of a full-length recombinant protein. Kinase inhibitors, which show a particular effect on NS5A phosphorylation in cells, can be retested in vitro on a particular kinase candidate. The problem with this approach is that purified components, like the purified NS5A substrate and the kinase of interest, are not always available.

Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

G-protein betagamma dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms beta and gamma. Recombinant Gbetagamma purified from Sf9 cells via metal-affinity and anion exchange chromatography exhibited a wortmannin-insensitive phospholipid kinase activity that copurified from the insect cells. To exclude false-positive results of Gbetagamma-dependent lipid kinase activity, the elimination of insect phospholipid kinase from Gbetagamma protein samples is necessary to avoid interference with the intrinsic lipid kinase activity of PI3K isoforms in reconstitution experiments. Here we describe an improved procedure of Gbeta(1)gamma(2) purification from cell membranes that separates the contaminating phospholipid kinase.

Selenophosphate synthetase (EC 2.7.9.3), the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide, for the synthesis of selenocysteine. The kinetoplastid Leishmania major and Trypanosoma brucei selD genes were cloned and the SELD protein overexpressed and purified to apparent homogeneity. The selD gene in L. major and T. brucei are respectively 1197 and 1179 bp long encoding proteins of 399 and 393 amino acids with molecular masses of 42.7 and 43 kDa. The molecular mass of 100 kDa for both (L. major and T. brucei) SELDs is consistent with dimeric proteins. The kinetoplastid selD complement Escherichia coli (WL400) selD deletion confirming it is a functional enzyme and the specific activity of these enzymes was determined. A conserved Cys residue was identified both by multiple sequence alignment as well as by functional complementation and activity assay of the mutant (Cys to Ala) forms of the SELD identifying this residue as essential for the catalytic function.

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

Selenophosphate synthetase (SPS) catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons. The crystal structure of an N-terminally (25 residues) truncated fragment of SPS (SPS-DeltaN) from Aquifex aeolicus has been determined at 2.0 A resolution. The structure revealed SPS to be a two-domain alpha/beta protein, with domain folds that are homologous to those of PurM-superfamily proteins. In the crystal, six monomers of SPS-DeltaN form a hexamer of 204 kDa, whereas the molecular weight estimated by ultracentrifugation was approximately 63 kDa, which is comparable to the calculated weight of the dimer (68 kDa).

The plant ethylene receptor ETR1, which shows substantial sequence homology to typical bacterial histidine kinases, is involved in the coordination of several growth and development processes. Fluorescence polarization studies presented here demonstrate a specific interaction of ETR1 with the histidine-containing transfer protein AHP1, supporting the idea that a phosphorelay module is involved in ethylene signaling. The sensitive assay employed in our studies allows analysis of protein-protein interactions in a homogenous aqueous environment, exact control of external parameters, and quantitative analysis of the affinity and stability of the complex. Thereby it provides the basics for a more quantitative elucidation of phosphorelay modules acquired in phytohormone signaling.

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the alpha-d-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (<5%) phosphopentomutase and phosphoglucomutase activities compared with PGM2, but was about 5-20-fold better than the latter enzyme in catalyzing the 1,3-bisphosphoglycerate-dependent synthesis of glucose 1,6-bisphosphate and other aldose-bisphosphates. Furthermore, quantitative real-time PCR analysis indicated that PGM2L1 was mainly expressed in brain where glucose-1,6-bisphosphate synthase activity was previously shown to be particularly high. We conclude that mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase correspond to two closely related proteins, PGM2 and PGM2L1, encoded by two genes that separated early in vertebrate evolution.

Lipid-derived inositol phosphates (IPs) are a complex group of second messengers generated by the sequential phosphorylation of inositol 1,4,5-trisphosphate (IP(3)). Synthetic pathways leading from IP(3) to the formation of inositol tetrakisphosphate IP(4), inositol pentakisphosphate IP(5), inositol hexakisphosphate IP(6), and inositol pyrophosphates PP-IPs have been elucidated in eukaryotes from yeast to human. Studies have attributed a variety of cellular functions to IPs, highlighting the importance of understanding how the pathways for their synthesis are regulated. This chapter summarizes experimental techniques for the biochemical characterization of the key inositol phosphate kinases IPKs necessary for producing the diverse array of IP species.

This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365-26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase.

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Two new papers (Kuranaga et al., 2006; Oshima et al., 2006) describe a previously uncharacterized Drosophila kinase (DmIKK epsilon) that regulates the abundance of DIAP1, a protein best known for its ability to inhibit apoptosis. However, DmIKK epsilon-mediated degradation of DIAP1 does not regulate apoptosis as might be predicted but instead regulates actin dynamics, cell morphology, and the differentiation of sensory organ precursor cells.

Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'-diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K (cat)/K (m), 4.3 x 10(4), found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

A protein of 75,000 Daltons with levoglucosan kinase activity was purified from Aspergillus niger. After in-gel digestion by trypsin, a 14-mer peptide was sequenced and analyzed by LC-ESI-MS/MS. Using a primer derived from the 14-mer peptide in combination with Oligo-(dT)18, a cDNA fragment was obtained by RT-PCR. A search of the GenBank database indicated that the protein had not been identified before. A similar protein named hypothetical protein FG07802.1 (EAA77996.1) was found to exist in Gibberella zeae by Blastx search. Using a primer derived from the protein, a cDNA fragment of second RT-PCR was cloned into plasmid pAJ401, which was transformed to Saccharomyces cerevisiae H158 and expressed. Two positive levoglucosan assimilating recombinants were selected. The lgk gene was screened and identified.

A new chromatography system, AKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at > 90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine- and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg2+. The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 A for the apoenzyme crystals and 1.93 A for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer.

In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688-695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec and, in addition, had a requirement for ATP and Mg2+. Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation., (Copyright 2004 The National Academy of Sciencs of the USA)