A fluorometric method for the assay of protein kinase activity.

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.

Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity.

Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr4+-phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL-1. Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL-1 with a detection limit (LOD) of 0.039 U mL-1. Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results.

Statin-related myotoxicity / Miotoxicidad por estatinas

Statin therapy has a very important role in decreasing cardiovascular risk, and treatment non-compliance may therefore be a concern in high cardiovascular risk patients. Myotoxicity is a frequent side effect of statin therapy and one of the main causes of statin discontinuation, which limits effective treatment of patients at risk of or with cardiovascular disease. Because of the high proportion of patients on statin treatment and the frequency of statin-related myotoxicity, this is a subject of concern in clinical practice. However, statin-related myotoxicity is probably underestimated because there is not a gold standard definition, and its diagnosis is challenging. Moreover, information about pathophysiology and optimal therapeutic options is scarce. Therefore, this paper reviews the knowledge about the definition, pathophysiology and predisposing conditions, diagnosis and management of statin-related myotoxicity, and provides a practical scheme for its management in clinical practice (AU)

El tratamiento con estatinas tiene un papel fundamental en la reducción del riesgo cardiovascular, y la falta de adherencia al mismo es motivo de preocupación en los pacientes con alto riesgo. La miotoxicidad es un efecto secundario frecuente y una de las principales causas de interrupción de las estatinas, lo que limita el tratamiento eficaz de los pacientes con riesgo de enfermedad cardiovascular. Considerando la elevada proporción de pacientes en tratamiento con estatinas, y la frecuencia de miotoxicidad asociada, este es un tema relevante en la práctica clínica. Sin embargo, la miotoxicidad por las estatinas está probablemente subestimada debido a que no hay una definición bien establecida y su diagnóstico resulta difícil. Además, la información sobre la fisiopatología y las opciones terapéuticas óptimas es escasa. En este artículo revisamos la definición, fisiopatología y condiciones predisponentes, diagnóstico y tratamiento de la miotoxicidad por las estatinas, y proponemos un esquema práctico para su manejo (AU)

Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

Development of LanthaScreen cellular assays for key components within the PI3K/AKT/mTOR pathway.

The PI3K/AKT/mTOR pathway is central to cell growth and survival, cell cycle regulation, and programmed cell death. Aberrant activation of this signaling cascade is linked to several disease states, and thus many components of the pathway are attractive targets for therapeutic intervention. However, the considerable degree of complexity, crosstalk, and feedback regulation that exists within the pathway (especially with respect to the regulation of mTOR and its complexes) underscores the need for a comprehensive set of cell-based assays to properly identify and characterize small-molecule modulators. Here, the development and application of time-resolved Förster resonance energy transfer (TR-FRET)-based assays to enable the phosphoprotein analysis of key pathway components in a cellular format are reported. The LanthaScreen cellular assay platform uses FRET between a terbium-labeled phosphorylation site-specific antibody and an expressed green fluorescent protein fusion of particular kinase substrate and provides an assay readout that is ratiometric, robust, and amenable to high-throughput screening applications. Assays specific for 5 different targets within the pathway are highlighted: Ser183 and Thr246 on the proline-rich AKT substrate 40 kDa (PRAS40), Ser457 on programmed cell death protein 4 (PDCD4), and Thr308 and Ser473 on AKT. Each assay was evaluated under various experimental conditions and individually optimized for performance. Known pathway agonists and a small panel of commercially available compounds were also used to complete the assay validation. Taken together, these data demonstrate the utility of a related set of cell-based assays to interrogate PI3K/AKT/mTOR signaling and provide a template for the development of similar assays for other targets.

Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense.

Solanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity.

A simple strategy for mitigating the effect of data variability on the identification of active chemotypes from high-throughput screening data.

Among the several goals of a high-throughput screening campaign is the identification of as many active chemotypes as possible for further evaluation. Often, however, the number of concentration response curves (e.g., IC(50)s or K(i)s) that can be collected following a primary screen is limited by practical constraints such as protein supply, screening workload, and so forth. One possible approach to this dilemma is to cluster the hits from the primary screen and sample only a few compounds from each cluster. This introduces the question as to how many compounds must be selected from a cluster to ensure that an active compound is identified, if it exists at all. This article seeks to address this question using a Monte Carlo simulation in which the dependence of the success of sampling is directly linked to screening data variability. Furthermore, the authors demonstrate that the use of replicated compounds in the screening collection can easily assess this variability and provide a priori guidance to the screener and chemist as to the extent of sampling required to maximize chemotype identification during the triage process. The individual steps of the Monte Carlo simulation provide insight into the correspondence between the percentage inhibition and eventual IC(50) curves.

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format.

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.

Real-time protein kinase assay.

We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

Impact of external calcium and calcium sensors on ginsenoside Rb1 biosynthesis by Panax notoginseng cells.

The effects of external calcium concentrations on biosynthesis of ginsenoside Rb1 and several calcium signal sensors were quantitatively investigated in suspension cultures of Panax notoginseng cells. It was observed that the synthesis of intracellular ginsenoside Rb1 in 3-day incubation was dependent on the medium Ca2+ concentration (0-13 mM). At an optimal Ca2+ concentration of 8 mM, a maximal ginsenoside Rb1 content of 1.88 +/- 0.03 mg g(-1) dry weight was reached, which was about 60% and 25% higher than that at Ca2+ concentrations of 0 and 3 mM, respectively. Ca2+ feeding experiments confirmed the Ca2+ concentration-dependent Rb1 biosynthesis. In order to understand the mechanism of the signal transduction from external Ca2+ to ginsenoside biosynthesis, the intracellular content of calcium and calmodulin (CaM), activities of calcium/calmodulin-dependent NAD kinase (CCDNK) and calcium-dependent protein kinase (CDPK), and activity of a new biosynthetic enzyme of ginsenoside Rb1, i.e., UDPG:ginsenoside Rd glucosyltransferase (UGRdGT), in the cultured cells were all analyzed. The intracellular calcium content and CCDNK activity were increased with an increase of external Ca2+ concentration within 0-13 mM. In contrast, the CaM content and activities of CDPK and UGRdGT reached their highest levels at 8 mM of initial Ca2+ concentration, which was also optimal to the ginsenoside Rb1 synthesis. A similar Ca2+ concentration-dependency of the intracellular contents of calcium and CaM and activities of CCDNK, CDPK, and UGRdGT was confirmed in Ca2+ feeding experiments. Finally, a possible model on the effect of external calcium on ginsenoside Rb1 biosynthesis via the signal transduction pathway of CaM, CDPK, and UGRdGT is proposed. Regulation of external Ca2+ concentration is considered a useful strategy for manipulating ginsenoside Rb1 biosynthesis by P. notoginseng cells.

A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases.

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.

The outer membrane of plant mitochondria contains a calcium-dependent protein kinase and multiple phosphoproteins.

Highly purified mitochondria from potato (Solanum tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross-contamination. When the matrix and inner membrane fractions were incubated with [gamma-32P]ATP only one and three prominent phosphoproteins were detected after SDS-PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, 18, 26, 43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca(2+)-dependent protein kinase and more than 20 endogenous substrates.

[Changes in the activity of 5'-nucleotidase and protein kinase of the hepatic plasma membrane depending on the dose rate in x-ray irradiation of rats]. / Izmenenie aktivnosti 5'-nukleotidazy i proteinkinazy plazmaticheskoi membrany pecheni v zavisimosti ot moshchnosti dozy pri rentgenovskom obluchenii krys.

In studying the effect of X-irradiation of rats with a dose of 4 Gy at different dose rates (96 and 4.6 cGy/min) the authors have revealed an increase in 5'-nucleotidase activity at a high dose rate (the first 24 h following irradiation) and a decrease in protein-kinase activity at a low dose rate (on days 3 and 7). The preinjection of alpha-tocopherol prevents the changes observed.

Expression of the Raf-1 protein in rat brain during development and its hormonal regulation in hypothalamus.

To study mechanisms involved in the sexual differentiation of the rat brain, the expression of the protein product of the proto-oncogene c-raf-1 (Raf-1) was examined. Biochemical and immunocytochemical analyses localized Raf-1 in embryonic rat brain regions and demonstrated hormonally induced changes in Raf-1 expression. For this study an affinity-purified anti-peptide antiserum specific for Raf-1 (NH-44) was used. Western blots revealed an approximately 77 kD polypeptide isolated in the cytosol of developing rat brains. Raf-1 levels were highest in the embryonic (E) day 22 female hypothalamus (HYP), and approximately twofold higher than levels detected in male HYP at E22 as determined by quantitative protein dot blot and semiquantitative Western blot analyses. Raf-1 levels in HYP were greater than those in either brain stem (BS) or cortex. Immunocytochemical analysis revealed high levels of Raf-1 in selective brain regions (e.g., the ventromedial nucleus in the HYP, the mitral cell layers in the main and accessory olfactory bulbs (OB), and the locus coeruleus) at E22 and postnatal (P) day 1. Lower levels of immunoreactivity were observed in many areas of the perinatal neuraxis. To test hormonal regulation of Raf-1, testosterone propionate (TP) was administered to pregnant rats on E17; male and female fetuses were examined on E22. This treatment significantly decreased Raf-1 levels in female HYP, but not in male HYP, as determined by Western blot analysis. No significant sex difference or response to prenatal hormone treatments were observed in either brain stem or cortex. No significant sex difference was noted postnatally, and administration of TP 3 h after birth did not change Raf-1 levels examined 24 h later. In summary, Raf-1 was localized within selective regions of the rat brain, and its expression was altered by exogenous prenatal hormonal stimulation. One role for Raf-1 in signal transduction may be to delimit hormonal critical periods in sexual differentiation of the brain.

Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey.

In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate. This suggests that the kinase may be differentially engaged in pre- and postsynaptic functions at certain synapses.

Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases.

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

Dot assay for neomycin phosphotransferase activity in crude cell extracts.

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.

[Level of cyclic AMP-dependent protein kinase isozyme in normal liver and hepatoma tissue and the effect of sodium selenite].

Sodium selenite in normal saline was administered intraperitoneally (1 mg/kg) into mice bearing ascitic hepatocarcinoma for 4 days. The cyclic AMP-dependent protein kinase isozymes (type I and type II) in normal liver and hepatocarcinoma cells were separated and assayed. The results show that the level of type I/II is markedly higher in hepatocarcinoma than in the normal liver cells. Sodium selenite is able to reduce it towards the normal level. Further analysis shows that the chief function of sodium selenite is to reduce the raised level of type I/II in hepatocarcinoma cells which, in fact, is due to the increase of total amount of type I cyclic AMP-dependent protein kinase. This paper presents the speculation that one of the mechanisms of the inhibitory effect of sodium selenite on carcinogenesis may be due to the selective action of this compound on the cyclic AMP-dependent protein kinase isozymes in tumor cells, thus inhibiting cancer cell division and facilitating differentiation and reversion.