Mitochondria generate cellular energy through oxidative phosphorylation and perturbation of this process can lead to increased levels of reactive oxygen species (ROS). It is critical that mitochondria communicate with the nucleus to regulate gene expression and maintain ROS homeostasis. This occurs via retrograde signalling pathways. Recently, a novel nuclear role was uncovered for Clk-1, a mitochondrial enzyme with a well-established role in oxidative phosphorylation. Upon conditions of increased ROS, a pool of Clk-1 translocates to the nucleus where it associates with chromatin and regulates gene expression. Experiments described in this thesis were aimed at characterising the regulation of Clk-1 localisation and dissecting the biological function of nuclear Clk-1.A non-classical nuclear localisation signal in the N-terminal region of Clk-1 was identified, containing three conserved Arginine residues, the most important of which was Arginine28. It was confirmed that the positive charge of Arginine is important for determining Clk-1 nuclear localisation. In addition, Tyrosine26 was also identified as having a regulatory role in Clk-1 localisation, possibly through a phosphorylation event. Increased ROS levels block the import of Clk-1 into the mitochondrial matrix and its cleavage by the mitochondrial processing peptidase. However, uncleaved Clk-1 was detected in the mitochondrial fraction, in addition to the nucleus, upon oxidative stress treatment. These results suggest that a pool of Clk-1 pre-protein may first be targeted to mitochondria prior to translocating to the nucleus.Interestingly it was found that a potentially misfolded Clk-1 mutant (E178K) is not ROS-responsive and that uncleaved Clk-1(E178K) predominantly associates with mitochondria. Similarly, a pharmacological inducer of Clk-1 misfolding promoted the association of uncleaved Clk-1 with the mitochondrial fraction. Collectively, these results suggest that Clk-1 import efficiency and the kinetics of Clk-1 folding determine its subcellular localisation.To specifically investigate the function of nuclear Clk-1, a conditional knock-in mouse model featuring a mutant version of Clk-1 (R28A) with impaired nuclear localisation is being generated. The cloning strategy employed to generate the targeting vector for the R28A conditional knock-in mouse and the strategy used to screen for positive mouse embryonic stem cell clones harbouring the R28A mutation in their genome, is presented in this thesis.The identification of Clk-1 as a novel regulator of mitochondrial to nuclear signalling has significant implications for understanding the cellular mechanisms that regulate mitochondrial homeostasis and could contribute to our understanding of diseases in which mitochondrial homeostasis has been disrupted.
|Date of Award||1 Aug 2015|
- The University of Manchester
|Supervisor||Alan Whitmarsh (Supervisor) & Claudia Wellbrock (Supervisor)|