Dunn, L.M.; (2011) Characterisation of leucine-rich repeat kinase-2 regulation and kinase function. Doctoral thesis, UCL (University College London).
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Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are one of the most common causes of genetic Parkinson’s disease (PD), with mutations thought to account for around 5% of all familial cases. LRRK2 is a large protein with a kinase and GTPase domain and multiple protein-‐protein interaction domains. Regulation of this protein is complex, with GTPase activity known to regulate kinase activity. Similarly, LRRK2 can autophosphorylate and is thought to form a dimer when active. Mutations in LRRK2 are numerous, with the most prevalent mutations occurring in the enzymatic core of this protein. This thesis describes work done to characterise the regulation and functioning of LRRK2, in order to further contribute towards understanding how mutations in this protein can lead to the pathogenesis of PD. Using BlueNative PAGE and glycerol gradient centrifugation, the quaternary structure of LRRK2 was assessed. In vitro kinase assays were used to characterise kinase activity of recombinant LRRK2 and a number of putative kinase substrates were also investigated. Identification of new kinase substrates was attempted and immunoprecipitation of LRRK2 to identify novel binding partners was also performed. Results of these experiments showed that familial mutations do not affect the ability of LRRK2 to form complexes. Instead, some mutations are affecting the enzymatic activity of LRRK2. Dephosphorylating LRRK2 showed that dimer formation is dependent on phosphorylation. Dephosphorylated forms of LRRK2 were more likely to be monomeric and displayed lower kinase activity than higher molecular weight forms. In vitro kinase assays to evaluate LRRK2 kinase substrates showed that α-synuclein is phosphorylated at low levels by G2019S but not wild-type LRRK2. Attempts to identify novel kinase substrates and binding partners of LRRK2 were unsuccessful, however evaluation of putative kinase substrates in vitro showed that DVL3 and TUBB5 may be good candidates for further investigation, as they were robustly phosphorylated by LRRK2. These results contribute towards our understanding of how LRRK2 functions and future studies based on these results may prove useful in aiding our understanding of how LRRK2 can cause PD pathogenesis.
|Title:||Characterisation of leucine-rich repeat kinase-2 regulation and kinase function|
|Open access status:||An open access version is available from UCL Discovery|
|Additional information:||Pages 215-218 comprises third party copyright material, which has been removed from the e-thesis.|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Neurology > Molecular Neuroscience|
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