Activation of the Parkinson's disease kinase LRRK2 by Rab GTPases

  • Elena Purlyte

Student thesis: Doctoral ThesisDoctor of Philosophy


Parkinson’s disease (PD) variants of the LRRK2 kinase increase the phosphorylation of its substrate Rab GTPases. Previous work indicated that Rab29, one of the LRRK2 substrates and a candidate gene for PD risk, might play an upstream role in LRRK2 kinase activity. It was shown that Rab29 overexpression in cells, together with LRRK2, enhances LRRK2 autophosphorylation and phosphorylation of its other substrate Rab GTPases. This effect is enhanced by the hyper-activating PD variants of LRRK2.

At first, I focused on the Rab29-dependent activation. I identified the key role of the LRRK2 N-terminal armadillo and ankyrin domains as well as the GTP-bound state of LRRK2 in the activation. Our collaborators have shown that Rab29 overexpression recruits cytosolic LRRK2 to the Golgi, where Rab29 resides, and that the membrane-localisation is key for LRRK2 activation. Due to these findings, I focused further on LRRK2 localisation in response to overexpression of Rab29 and other Rab GTPases. This work revealed that Rab29, as well as Rab12 and two non-LRRK2 substrates Rab32 and Rab38, are able to recruit overexpressed LRRK2 via the N-terminus of LRRK2, while a group of other LRRK2 substrate Rab GTPases (Rab8a/b, Rab10, Rab35, Rab43) recruit LRRK2 independent of its N-terminus. I narrowed down the minimal N-terminal fragment of LRRK2 to a 150 amino acid region within the armadillo domain that is sufficient to be recruited by Rab29 and I investigated potential key residues necessary for the interaction.
For another project, I investigated the role of 98 variants of LRRK2 identified in PD patients on LRRK2 cellular functions. Together with A. Kalogeropulou and Dr F. Tonelli, I evaluated the LRRK2 variant phosphorylation of endogenous Rab10 in cells and phosphorylation of LRRK2 biomarker sites. With the help of Dr Alan Prescott from the Dundee Imaging Facility I also evaluated the effect of the LRRK2 variants on recruitment to the Golgi by Rab29 and ability to localise to the microtubules under inhibitor treatment. Through this work we have identified multiple variants that were previously unknown to hyper-activate LRRK2, as well as some that decreased LRRK2 kinase activity in cells. None of the variants were able to disrupt LRRK2 co-localisation with Rab29, however, I have identified variants that disrupted microtubule localisation of LRRK2. My preliminary data has shown lack of a link between LRRK2 kinase activity in vivo and microtubule co-localisation and even though most of the known pathogenic variants of LRRK2 had high phosphorylation of Rab10, they had varied levels of association with microtubules suggesting that microtubule association is not a reliable marker for variant pathogenicity.

For my final project, in collaboration with Prof. A. Khan’s group I investigated the interaction between one of the LRRK2 substrates Rab8a and its phospho-specific interactor RILPL2. I have optimised an assay to produce large quantities of Rab8a phosphorylated at the LRRK2 site using a promiscuous kinase MST3. This phosphorylated Rab8a was used by Prof. A. Khan to determine the crystal structure of the complex of phosphorylated Rab8a and RILPL2 RH2 domain. I performed co-immunoprecipitation experiments using multiple mutants of RILPL2 RH2 domain to verify the key residues for complex formation.

Together, this work has provided important knowledge that can be utilised for further study of the LRRK2 and Rab GTPase pathways – as well as to aid selection of PD patients with LRRK2 variants that would most likely benefit from LRRK2 inhibitor treatments in clinical trials.
Date of Award2021
Original languageEnglish
SupervisorDario Alessi (Supervisor)


  • LRRK2
  • Parkinson's disease
  • Rab GTPases
  • RILPL2
  • Rab29
  • Rab8a
  • phosphorylation

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