A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms

Shared decision making for psychiatric medication management: beyond the micro-social

Background: Mental health care has lagged behind other health-care domains in developing and applying shared decision making (SDM) for treatment decisions. This is despite compatibilities with ideals of modern mental health care such as self-management and recovery-oriented practice, and growing policy-level interest. Psychiatric medication is a mainstay of mental health treatment, but there are known problems with prescribing practices, and service users report feeling uninvolved in medication decisions and concerned about adverse effects. SDM has potential to produce better tailoring of psychiatric medication to individuals' needs. Objectives: This conceptual review argues that several aspects of mental health care that differ from other health-care contexts (e.g. forms of coercion, questions about service users' insight and disempowerment) may impact on processes and possibilities for SDM. It is therefore problematic to uncritically import models of SDM developed in other health-care contexts. We argue that decision making for psychiatric medication is better understood in a broader way that moves beyond the micro-social focus of a medical consultation. Contextualizing specific medication-related consultations within longer term relationships, and broader service systems enables recognition of the multiple processes, actors and agendas that shape how psychiatric medication is prescribed, managed and used, and which may facilitate or impede SDM. Conclusion: A broad conceptualization of decision making for psychiatric medication that moves beyond the micro-social can account for why SDM in this domain remains a rarity. It has both conceptual and practical utility for evaluating research evidence, identifying future research priorities and highlighting fruitful ways of developing and implementing SDM in mental health care

The phenomenology of empowerment in collective action

Recent research has hypothesized that empowerment can arise from collective action through collective self-objectification (CSO), defined as action that actualizes participants' social identity against the power of dominant groups. Activists (N=37) described several experiences that made them feel empowered (and disempowered). Among the various explanations they offered for these feelings, the most prominent were CSO, unity, and support (or their absence). CSO was also predictive of reports of positive emotion, although unity was the best predictor of reports of further involvement. Overall, the study suggests that actualizing one's social identity through collective action has personal as well as political significance

Characterization of NLP, a novel centrosomal substrate of the NEK2 kinase

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterization of NLP, a novel centrosomal substrate of the NEK2 kinase

The centrosome is a non-membraneous organelle composed of two centrioles surrounded by pericentrolar material. The primary function of the centrosome is to act as the dominant microtubule organizing centre in animal cells. It therefore contributes to formation of both the interphase cytoskeleton and bipolar mitotic spindle. Both centrosome structure and microtubule organization are controlled in a cell cycle dependant manner by protein phosphorylation. The centrosomal kinase Nek2 regulates centrosome organization, mitotic progression and bipolar spindle assembly. However, the only core centrosomal substrate of this kinase so far identified is C-Napl, a structural protein required for centriolar cohesion. The aims of this project were therefore to isolate a Xenopus laevis homologue of C-Napl in order to study its function using Xenopus based cell free assays and identify and characterize novel centrosomal substrates of Nek2. By database screening, we identified a Xenopus protein of high similarity to C-Napl called rootletin, while using the yeast two hybrid system we identified the Xenopus Nip protein as a novel substrate of Nek2. Human Nip is a recently characterized centrosomal protein involved in microtubule organization and which is regulated by another centrosomal kinase, Plkl. Antibodies were raised to Xenopus Nip and used to confirm subcellular localization to the centrosome in Xenopus cells. Further localization and expression studies revealed that Nip is a mother centriole specific protein that is displaced from the centrosome, but not degraded, during mitosis. These data suggest that Nip is involved in interphasic microtubule anchorage. By transfection into Xenopus and human cells, we found that although Nek2 and Plkl phosphorylate Nip at distinct sites, they can both trigger Nip displacement from the centrosome at the onset of mitosis. Finally, data were obtained raising the possibility that Nek2 may act as a novel priming kinase for recruitment of Plkl to its substrate Nip

A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation.

mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms

mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry

Variants in the IMP2 (insulin-like growth factor 2 [IGF2] mRNA-binding protein 2) gene are implicated in susceptibility to type 2 diabetes. We describe the ability of mammalian target of rapamycin (mTOR) to regulate the cap-independent translation of IGF2 mRNA through phosphorylation of IMP2, an oncofetal RNA-binding protein. IMP2 is doubly phosphorylated in a rapamycin-inhibitable, amino acid-dependent manner in cells and by mTOR in vitro. Double phosphorylation promotes IMP2 binding to the IGF2 leader 3 mRNA 5′ untranslated region, and the translational initiation of this mRNA through eIF-4E- and 5′ cap-independent internal ribosomal entry. Unexpectedly, the interaction of IMP2 with mTOR complex 1 occurs through mTOR itself rather than through raptor. Whereas depletion of mTOR strongly inhibits IMP2 phosphorylation in cells, comparable depletion of raptor has no effect; moreover, the ability of mTOR to phosphorylate IMP2 in vitro is unaffected by the elimination of raptor. Dual phosphorylation of IMP2 at the mTOR sites is evident in the mouse embryo, likely coupling nutrient sufficiency to IGF2 expression and fetal growth. Doubly phosphorylated IMP2 is also widely expressed in adult tissues, including islets of Langerhans

High-throughput image-based screens for genes regulating the phosphorylation of rpS6.

<p>A. Immunofluorescence analysis (IF) of rpS6Ser(235/236) phosphorylation. Mia-Paca 2 cells were transfected in 384-well plates with a control, nonspecific RNAis NS1 (upper panel) and (NS2) (lower panel), and RNAi pools directed at S6K, TOR and Raptor, TSC1, TSC2 and PTEN. After 72 hours they were fixed, permeabilized and stained by using a rabbit monoclonal anti-S6-P(Ser 235/236) primary antibody, detected with secondary anti-rabbit Alexa 488 antibody (green). Nuclei are stained with DAPI (blue). Representative images are shown. B. Quantitation of cytoplasmic S6-P levels. The bars indicate the % of total MIA PaCa-2 cells (estimated by nuclear count) that exhibit cytoplasmic S6-P immunofluorescence at an intensity above an arbitrary threshold (% S6-P positive cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#sec002" target="_blank">methods</a>). The z’ is 0.31 for the combined use of NS1 and NS2; because NS2 gave consistently higher z’ than NS1 (e.g., 0.45 vs 0.32 for the experiment shown) NS2 was used exclusively in the primary screen; error bars represent 1S.D. * = p<0.01. C. Flow chart of the primary screen: Summary of the screening, hit analysis and hit selection. 21,121 genes were tested using RNAis composed of pools of 4 RNAi oligos (Dharmacon Library); 72 384-well plates were screened in triplicate (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.t001" target="_blank">Table 1</a>). The criteria for a “primary positive” are described in the text (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.t002" target="_blank">Table 2</a> lists genes not scored due to severe inhibition of proliferation). D. Results of the confirmation screen. From the 1046 “primary positives”, 870 genes, including all 161 positive kinases and the top 709 ranked by Q (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s005" target="_blank">S3 Table</a>), were examined in a confirmation screen wherein each of the four RNAis was tested individually. The pie chart indicates how many of the potential positive hits were confirmed by 0–4 individual siRNAs (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116096#pone.0116096.s006" target="_blank">S4 Table</a>).</p

siRNA-mediated depletion of QARS inhibits mTORC1 signaling.

<p>A. The effect of mTOR, QARS and LARS siRNA upon polypeptide knockdown and S6K-Thr³⁸⁹ phosphorylation. U2OS and Hela cells were transfected with siRNA oligos against mTOR, QARS (Q1,Q2), LARS(L1,L2) and a scramble control. After 72h amino acids were withdrawn for 2 hours and added back for 15 minutes as indicated. Cells extracts were subjected to SDS-PAGE and membranes were immunoblotted with the antibodies as indicated. The bar graphs display the combined results of three experiments (mean-/+ 1 S.D.; ** = p<0.0001 and * = p<0.002 vs scramble) The experiment shown in the bottom panels compares the effects of Thapsigargin (10M) with the indicated siRNAs on S6K-P (fourth from top), eIF2α(Ser51-P) (third from bottom) and the abundance of REDD1 (bottom). B. siRNA-mediated depletion of QARS inhibits global protein synthesis. Graphical representation of the combined results from three experiments (mean-/+ 1 S.D.) examining the effect, relative to scramble siRNA, of siRNA against mTOR, QARS and LARS on the abundance of the target polypeptides (upper), the relative phosphorylation of S6K-P (middle; ** = p<0.0001 and * = p<0.002 vs scramble) and on overall protein synthesis (bottom) in nutrient and serum replete U2OS cells. Analyses were carried out three days after transfection. ³⁵S[Methionine+cysteine] was added two hours before harvest; cycloheximide (CHX, 100μM) or carrier was added 30′ prior to ³⁵S. C. The effect of inhibiting translation for three days on mTORC1 signaling. Graphical representation of a dose response of cycloheximide (CHX) on global protein synthesis in U2OS cells. U2OS cells were plated in DMEM with 10% FCS +/- CHX and fresh media containing carrier or CHX was added every 24 hours for 72 hours. The cell were harvested at 72 hours; protein content expressed as a fraction of carrier control is plotted in the upper graph. Immunoblots of cell extracts for the proteins indicated are shown in the middle and the ratio of S6K-P/S6K (mean-/+ 1 S.D.) is shown in the bar graph at the bottom.</p