Sancho,, Rosa Maria Moreira; (2009) Identification and characterisation of the interaction between lrrk2 and dvl proteins. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Mutations in the cytoplasmic protein kinase dardarin (LRRK2) are the most common known cause of Parkinson's disease. LRRK2 is a cytosolic kinase that belongs to the ROCO family of proteins which are characterised by a Roc (Ras in complex proteins) domain with intrinsic GTPase activity and a COR (C-terminal of Roc) domain. The Roc-COR tandem domain controls LRRK2 kinase activity via an intramolecular process. The modification of LRRK2 GTPase and kinase activity by familial Parkinson's disease mutations in the Roc, COR and kinase domains is believed to lead to neuronal cell death but the pathways involved remain elusive. To improve our understanding of LRRK2 signalling pathways leading to cell death, this work focused on the identification and characterisation of LRRK2-interacting proteins. Using the yeast two-hybrid system, I found that the Roc-COR domain interacts with a family of phosphoproteins, the Dishevelled (DVL) family. DVL proteins were considered promising candidates for further analysis since they are key regulators of Wnt (Wingless/Int) signalling pathways leading to multiple downstream effects, including the activation of small GTPases, such as Racl, that are structurally similar to the Roc domain. The interaction of LRRK2 and DVL proteins was characterised in further detail using the yeast two-hybrid system, biochemical and mammalian cellular models. The binding domains of LRRK2 and DVL mediating the interaction were determined and LRRK2 was shown to re-distribute to DVL aggregates upon co-expression in cellular models. Moreover, I found that LRRK2 mutations segregating with Parkinson's disease influence the interaction with DVLl. Lastly, using quantitative RT-PCR and immunohistochemistry I established that LRRK2 and DVLl-3 transcripts and proteins were expressed in human brain, including in the substantia nigra.

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