A rapid method for determining protein kinase phosphorylation specificity

Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred peptide phosphorylation motif for a given kinase permits the production of efficient peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial peptide library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Metabolic Stress Controls mTORC1 Lysosomal Localization and Dimerization by Regulating the TTT-RUVBL1/2 Complex

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Highlights? Glucose and glutamine feed back to promote mTORC1 signaling through ATP production ? Energetic stress prevents mTOR lysosomal localization independently of AMPK and Rag ? ATP-dependent TTT-RUVBL complex is disassembled and repressed by energetic stress ? TTT-RUVBL is required for mTORC1 functional assembly and lysosomal localization     

Development of a transient large strain contact method for biological heart valve simulations

A new 2D method to implement transient contact using Comsol Multiphysics (finite element analysis software that enables multiphysics simulations) is described, which is based on Hertzian contact. This method is compared to the existing (default) contact method that does not enable real transient simulations, but instead performs steady-state solutions where time is a constant. The two types of contact modelling have been applied to simple 2D biological heart valve models, undergoing strains in the region of 10% under 20 kPa pressure (applied over 0.3 s). Both the methods predicted comparable stress patterns, locations of peak stresses, deformations and directions of principal stress. The default contact method predicted slightly greater contact stresses, but spreads over a shorter surface length than the new contact method. The default contact method is useful for contact systems with little transient dependency, due to ease of use. However, where transient conditions are important the new contact method is preferred.     

Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in?vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.     

Wavelet-based Gaussian-mixture hidden Markov model for the detection of multistage seizure dynamics: A proof-of-concept study

Background  

Epilepsy is a common neurological disorder characterized by recurrent electrophysiological activities, known as seizures. Without the appropriate detection strategies, these seizure episodes can dramatically affect the quality of life for those afflicted. The rationale of this study is to develop an unsupervised algorithm for the detection of seizure states so that it may be implemented along with potential intervention strategies.     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing