A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.     

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.     

High-content siRNA screen of the kinome identifies kinases involved in Git2-induced mesenchymal-epithelial transition

Epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) programs are involced in the metastatic process. More and more evidence confirms that EMT is vital for the initiation and dissemination of cancer cells whereas MET is critical for successful metastatic colonization of a secondary organ. The regulating mechanism of EMT mediated cancer progression and metastasis has been deeply investigated. However, what processes are dependent on MET in metastatic cascades remains unclear. Here, we created a cell based high-content siRNA screen using the breast cancer cell line 4TO7 to search for kinases that were involved in Git2-induced MET. Our results revealed that 58 kinases including transferase, phosphorylation regulators, ATP/nucleotide partners potentially participate in Git2-induced MET. Our preliminary data is expected to facilitate elucidation of the mechanism on how MET is initiated during cancer metastasis.     

A screen for genes that influence fibroblast growth factor signal transduction in Drosophila

The misexpression of an activated form of the FGF receptor (FGFR) Breathless in conjunction with downstream-of-FGF-receptor (Dof), an essential signaling molecule of the FGF pathway, in the Drosophila eye imaginal discs impairs eye development and results in a rough eye phenotype. We used this phenotype in a gain-of-function screen to search for modifiers of FGF signaling. We identified 50 EP stocks with insertions defining at least 35 genes that affect the rough eye phenotype. Among these genes, 4 appear to be specific for FGFR signaling, but most of the genes also influence other signaling pathways, as assessed by their effects on rough eyes induced by other activated receptor tyrosine kinases (RTKs). Analysis of loss-of-function alleles of a number of these genes in embryos indicates that in many cases the products are provided maternally and are involved in germ cell development. At least two of the genes, sar1 and robo2, show a genetic interaction with a hypomorphic dof allele, suggesting that they participate in FGF-mediated morphogenetic events during embryogenesis.     

Chimeric YACs were generated at unreduced rates in conditions that suppress coligation.

Chimerism is a major limitation of current YAC libraries. A method based on partially filled-in ends of restriction fragments was designed to avoid coligation as a possible source of chimeras. Model experiments using plasmid DNA as an insert showed that coligation was clearly avoided by this method. Pilot collections of YACs with an average insert size of 650kb were then constructed with and without the partial fill-in treatment. Starting from a mixture of a equal amounts of human and mouse DNA, none of 108 clones was positive by hybridization with both Alu and B2 probes, again suggesting that coligation was effectively blocked. However, 4 out of 10 clones still hybridized to 2 or more locations by FISH on chromosomes in human metaphase spreads, level similar to that in the clones made without the partial fill-in step. These results strongly suggest that chimeric clones generally arise by a mechanism independent of coligation, presumptively based on recombination.     

Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.     

Determination of optimal non-denaturing elution conditions from affinity columns by a solid-phase screen

The purification of biological macromolecules by affinity chromatography is a widespread technique used to separate a protein from other biological components. However, this method may destroy the protein's physiological activity because elution conditions aimed to dissociate the protein of interest from the high-affinity matrix often irreversibly denature it. In the present work, we have developed a solid-phase assay to determine the optimal elution conditions for any buffer (in two steps) by determining (i) the lowest buffer concentration yielding maximum dissociation from the immobilized component and (ii) the highest buffer concentration that can be used without the loss of the protein's binding activity. Any buffer that can be reasonably used between these defined concentrations is suitable for elution within this interval. The screen is easily performed within a few hours and only requires nanograms to a few micrograms of protein. As an example, we demonstrate that more than 95% of the human transferrin receptor bound to a transferrin-sepharose ligand affinity column can be eluted with full binding activity at KSCN concentrations between 232 and 414 nM, whereas elution with urea is not suitable to purify fully functional protein.     

A screen for genes that function in leg disc regeneration in Drosophila melanogaster

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported here aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. We screened a collection of 97 recessive lethal P-lacZ enhancer trap lines for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, we identified six genes (Krüppel-homolog-1, rpd3, jing, combgap, Aly and S6 kinase) that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration.     

A differential drug screen for compounds that select against antibiotic resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus "inverting" selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets.     

A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor

Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.     

An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye

We screened for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, we identified 46 lines, corresponding to insertions in 32 different loci, that elicited a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation.     

Nerve growth factor activates kinases that phosphorylate atypical protein kinase C

Activation of atypical protein kinase C by nerve growth factor (NGF) involves phosphorylation. In order to identify kinases that regulate atypical PKC (aPKC), we surveyed PC12 cell lysates for protein kinases that are activated by NGF and which could phosphorylate aPKC. Employing an in-gel kinase assay where aPKC-zeta was copolymerized within the gel matrix as a substrate, three kinases, pp175, pp87 and pp60, were identified as enzymes that phosphorylated aPKC. Phosphorylation of aPKC by these three kinases coincided with NGF-induced activation of the enzyme. Each kinase possessed a unique subcellular distribution pattern and could be activated by either ceramide or H(2)0(2), second messengers that mimic NGF signaling events. Upstream, pp175 and pp60 lie in a ras pathway, whereas pp87 lies in a pathway dependent upon src. Altogether, these findings reveal that the aPKCs are subject to regulation by a novel group of kinases.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.     

A genome-wide screen for essential yeast genes that affect telomere length maintenance

Telomeres are structures composed of repetitive DNA and proteins that protect the chromosomal ends in eukaryotic cells from fusion or degradation, thus contributing to genomic stability. Although telomere length varies between species, in all organisms studied telomere length appears to be controlled by a dynamic equilibrium between elongating mechanisms (mainly addition of repeats by the enzyme telomerase) and nucleases that shorten the telomeric sequences. Two previous studies have analyzed a collection of yeast deletion strains (deleted for nonessential genes) and found over 270 genes that affect telomere length (Telomere Length Maintenance or TLM genes). Here we complete the list of TLM by analyzing a collection of strains carrying hypomorphic alleles of most essential genes (DAmP collection). We identify 87 essential genes that affect telomere length in yeast. These genes interact with the nonessential TLM genes in a significant manner, and provide new insights on the mechanisms involved in telomere length maintenance. The newly identified genes span a variety of cellular processes, including protein degradation, pre-mRNA splicing and DNA replication.     

A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis

We introduce an approach for detection of drug-protein interactions that combines a new yeast three-hybrid screening for identification of interactions with affinity chromatography for their unambiguous validation. We applied the methodology to the profiling of clinically approved drugs, resulting in the identification of previously known and unknown drug-protein interactions. In particular, we were able to identify off-targets for erlotinib and atorvastatin, as well as an enzyme target for the anti-inflammatory drug sulfasalazine. We demonstrate that sulfasalazine and its metabolites, sulfapyridine and mesalamine, are inhibitors of the enzyme catalyzing the final step in the biosynthesis of the cofactor tetrahydrobiopterin. The interference with tetrahydrobiopterin metabolism provides an explanation for some of the beneficial and deleterious properties of sulfasalazine and furthermore suggests new and improved therapies for the drug. This work thus establishes a powerful approach for drug profiling and provides new insights in the mechanism of action of clinically approved drugs.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.     

A comprehensive update of the sequence and structure classification of kinases

Background  

A comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme.