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.
Amino acid regulation of TOR complex 1 AVRUCH, Joseph; XIAOMENG LONG; ORTIZ-VEGA, Sara ...
American journal of physiology: endocrinology and metabolism,
2009, Letnik:
59, Številka:
4
Journal Article
CARMA1 is a central regulator of NF- Kappa B activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF- Kappa B signaling downstream of the T-cell receptor (TCR). Computational ...analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca super(2+)/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF- Kappa B activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF- Kappa B activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF- Kappa B activation.
CARMA1 is a central regulator of NF-kappaB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-kappaB signaling downstream of the T-cell receptor (TCR). Computational ...analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca(2+)/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-kappaB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-kappaB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-kappaB activation.
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.
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.
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.
Early rehabilitation and mobilisation encompass patient-tailored interventions, delivered within intensive care, but there are few studies in children and young people within paediatric intensive ...care units.
To explore how healthcare professionals currently practise early rehabilitation and mobilisation using qualitative and quantitative approaches; co-design the Paediatric Early Rehabilitation and Mobilisation during InTensive care manual of early rehabilitation and mobilisation interventions, with primary and secondary patient-centred outcomes; explore feasibility and acceptability of implementing the Paediatric Early Rehabilitation and Mobilisation during InTensive care manual within three paediatric intensive care units.
Mixed-methods feasibility with five interlinked studies (scoping review, survey, observational study, codesign workshops, feasibility study) in three phases.
United Kingdom paediatric intensive care units.
Children and young people aged 0-16 years remaining within paediatric intensive care on day 3, their parents/guardians and healthcare professionals.
In Phase 3, unit-wide implementation of manualised early rehabilitation and mobilisation.
Phase 1 observational study: prevalence of any early rehabilitation and mobilisation on day 3. Phase 3 feasibility study: acceptability of early rehabilitation and mobilisation intervention; adverse events; acceptability of study design; acceptability of outcome measures.
Searched Excerpta Medica Database, Cumulative Index to Nursing and Allied Health Literature, MEDLINE, PEDro, Open grey and Cochrane CENTRAL databases.
Narrative synthesis.
In the scoping review we identified 36 full-text reports evaluating rehabilitation initiated within 7 days of paediatric intensive care unit admission, outlining non-mobility and mobility early rehabilitation and mobilisation interventions from 24 to 72 hours and delivered twice daily. With the survey, 124/191 (65%) responded from 26/29 (90%) United Kingdom paediatric intensive care units; the majority considered early rehabilitation and mobilisation a priority. The observational study followed 169 patients from 15 units; prevalence of any early rehabilitation and mobilisation on day 3 was 95.3%. We then developed a manualised early rehabilitation and mobilisation intervention informed by current evidence, experience and theory. All three sites implemented the Paediatric Early Rehabilitation and Mobilisation during InTensive care manual successfully, recruited to target (30 patients recruited) and followed up the patients until day 30 or discharge; 21/30 parents consented to complete additional outcome measures.
The findings represent the views of National Health Service staff but may not be generalisable. We were unable to conduct workshops and interviews with children, young people and parents to support the Paediatric Early Rehabilitation and Mobilisation during InTensive care manual development due to pandemic restrictions.
A randomised controlled trial is recommended to assess the effectiveness of the manualised early rehabilitation and mobilisation intervention.
A definitive cluster randomised trial of early rehabilitation and mobilisation in paediatric intensive care requires selection of outcome measure and health economic evaluation.
The study is registered as PROSPERO CRD42019151050. The Phase 1 observational study is registered Clinicaltrials.gov NCT04110938 (Phase 1) (registered 1 October 2019) and the Phase 3 feasibility study is registered NCT04909762 (Phase 3) (registered 2 June 2021).
This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 17/21/06) and is published in full in
; Vol. 27, No. 27. See the NIHR Funding and Awards website for further award information.
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.
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