Publications by authors named "Emma Hiley"

3 Publications

  • Page 1 of 1

Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.

Biochem J 2009 Apr;419(1):65-73

Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK.

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.
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http://dx.doi.org/10.1042/BJ20081646DOI Listing
April 2009

The Galpha12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans.

EMBO J 2006 Dec 30;25(24):5884-95. Epub 2006 Nov 30.

MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, University College London, Gower Sttreet, London, UK.

In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Galpha12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. We showed previously that RHO-1 stimulates ACh release by two separate pathways-one that requires UNC-13 and a second that does not. We show here that a non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons.
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http://dx.doi.org/10.1038/sj.emboj.7601458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698903PMC
December 2006

Rho is a presynaptic activator of neurotransmitter release at pre-existing synapses in C. elegans.

Genes Dev 2006 Jan;20(1):65-76

MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Pharmacology, University College, London WC1E 6BT, United Kingdom.

Rho GTPases have important roles in neuronal development, but their function in adult neurons is less well understood. We demonstrate that presynaptic changes in Rho activity at Caenorhabditis elegans neuromuscular junctions can radically change animal behavior via modulation of two separate pathways. In one, presynaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho-induced changes in neuronal morphology or synapse number; thus, Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterized mutation that severely reduces binding of RhoA to DAG kinase. Thus, a role for RhoA in controlling DAG levels is likely to have been underestimated.
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http://dx.doi.org/10.1101/gad.359706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1356101PMC
January 2006