A novel nuclear role for the mitochondrial hydroxylase Clk-1

Abstract

The lipid hydroxylase Clk-1 catalyses an essential step in the mitochondrialocalisedubiquinone biosynthetic pathway that is conserved throughout eukarya.Like many canonical mitochondrial proteins, murine Clk-1 is targeted andimported into mitochondria by virtue of a ~4kDa N-terminal mitochondrialtargeting domain that is cleaved following translocation into the mitochondrialmatrix. Clk-1 mutants in C. elegans and heterozygous Clk-1+/- mice exhibitincreased longevity and delayed development rates compared to wild typeindividuals, with associated changes in oxidative stress signalling pathways.Previous work in the laboratory identified human Clk-1 as a potential interactor ofSin1, 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-1is identified as a potential substrate of the Sin1-associated kinases cAMPdependentkinase, protein kinase C and unc-51-like kinase 1. Interestingly, afraction of Clk-1 was observed residing in the nucleus, in addition to itsmitochondrial localisation. The sequence determinants for Clk-1 nuclearlocalisation were found to be in the same N-terminal region required formitochondrial localisation and a single point mutant was identified thattranslocated to the mitochondria but not the nucleus. Oxidative stress treatmentwas shown to increase the level of uncleaved Clk-1 and this form was enriched innuclear and chromatin fractions. Clk-1 was found to be associated with over 1000genomic loci following Clk-1 chromatin immunoprecipitation followed by promotermicroarray analysis.Cells stably expressing the Clk-1 non-nuclear point mutant displayed decreasedresistance to oxidative stress-induced cell death and increased levels of oxidativespecies following treatment with exogenous stress. In addition, c-Jun N-terminalkinase signaling was enhanced in these cells in response to tumour necrosisfactor-alpha stimulation. Microarray analysis of these cells showed both positive andnegative transcript changes compared to wild type Clk-1 expressing cells whichwas significant for over 2000 genes. Functional clustering analysis identifiedenrichment for gene groups associated with glycolytic and tricarboxylic acid cyclemetabolism, Wnt signaling, and several specific differentiation and oncogenicpathways. Many of the genes identified are reported to be regulated by promotermethylation, and this was confirmed for glutathione-S-transferase P1 that hadsignificantly decreased expression in mutant Clk-1 expressing cells. Loss of Clk-1nuclearisation or Clk-1 activity within the nucleus could therefore be acting aspart of specific differentiation pathways either during early development orfollowing differentiation of specific cell lineages. Nuclear Clk-1's ties to respirationand cell survival pathways, and sensitivity to oxidative stress, could also implicateit in oncogenic progression and/or the Clk-1 ageing phenotype.

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alan Whitmarsh (Supervisor)