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Title: A novel nuclear role for the mitochondrial hydroxylase Clk-1
Author: Monaghan, Richard
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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The lipid hydroxylase Clk-1 catalyses an essential step in the mitochondria localised ubiquinone biosynthetic pathway that is conserved throughout eukarya. Like many canonical mitochondrial proteins, murine Clk-1 is targeted and imported into mitochondria by virtue of a ~4kDa N-terminal mitochondrial targeting domain that is cleaved following translocation into the mitochondrial matrix. Clk-1 mutants in C. elegans and heterozygous Clk-1+/- mice exhibit increased longevity and delayed development rates compared to wild type individuals, with associated changes in oxidative stress signalling pathways. Previous work in the laboratory identified human Clk-1 as a potential interactor of Sin1, a component of the mammalian target of rapamycin signaling pathway, which has also been associated with modulations that affect lifespan. Here the interaction between Clk-1 and Sin1 is further characterised and Clk-1 is identified as a potential substrate of the Sin1-associated kinases cAMP dependent kinase, protein kinase C and unc-51-like kinase 1. Interestingly, a fraction of Clk-1 was observed residing in the nucleus, in addition to its mitochondrial localisation. The sequence determinants for Clk-1 nuclear localisation were found to be in the same N-terminal region required for mitochondrial localisation and a single point mutant was identified that translocated to the mitochondria but not the nucleus. Oxidative stress treatment was shown to increase the level of uncleaved Clk-1 and this form was enriched in nuclear and chromatin fractions. Clk-1 was found to be associated with over 1000 genomic loci following Clk-1 chromatin immunoprecipitation followed by promoter microarray analysis. Cells stably expressing the Clk-1 non-nuclear point mutant displayed decreased resistance to oxidative stress-induced cell death and increased levels of oxidative species following treatment with exogenous stress. In addition, c-Jun N-terminal kinase signaling was enhanced in these cells in response to tumour necrosis factor-α stimulation. Microarray analysis of these cells showed both positive and negative transcript changes compared to wild type Clk-1 expressing cells which was significant for over 2000 genes. Functional clustering analysis identified enrichment for gene groups associated with glycolytic and tricarboxylic acid cycle metabolism, Wnt signaling, and several specific differentiation and oncogenic pathways. Many of the genes identified are reported to be regulated by promoter methylation, and this was confirmed for glutathione-S-transferase P1 that had significantly decreased expression in mutant Clk-1 expressing cells. Loss of Clk-1 nuclearisation or Clk-1 activity within the nucleus could therefore be acting as part of specific differentiation pathways either during early development or following differentiation of specific cell lineages. Nuclear Clk-1’s ties to respiration and cell survival pathways, and sensitivity to oxidative stress, could also implicate it in oncogenic progression and/or the Clk-1 ageing phenotype.
Supervisor: Whitmarsh, Alan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available