MRC-PPU researchers uncover potential cross-talk between signalling pathways linked to Parkinson’s disease

Researchers from the MRC-PPU have uncovered potential cross-talk between PINK1 and LRRK2 signalling pathways linked to Parkinson’s disease. This work, led by Professor Miratul Muqit and his team, was published this week in the Biochemical Journal.

Professor Muqit explains the background, research and outcomes from the study, "Mutations in genes encoding PINK1 (PTEN-induced kinase 1) are a leading cause of autosomal recessive early-onset Parkinson’s disease. PINK1 is a protein kinase that is activated upon mitochondrial damage to phosphorylate Ubiquitin and Parkin to stimulate Parkin E3 ligase activity and is a master-regulator of mitochondrial quality control. In 2015 Yu-Chiang Lai and Chandana Kondapalli of the Muqit lab found that PINK1 can also induce phosphorylation of a subset of Rab GTPases including Rab8A and Rab1B at a highly conserved Serine111 residue via an unknown intermediate kinase. This site is distinct from the Switch II Thr/Ser site that is phosphorylated by LRRK2 in which autosomal dominant mutations cause Parkinson’s.

"Ser111 lies within the SF3 domain of Rabs and PhD student Katie Mulholland teamed up with post-doc, Sophie Vieweg, of the Itzen lab at Technical University Munich (TUM) and later UKE Hamburg to investigate the consequence of phosphorylation. Using gene codon expansion technologies, Sophie expressed preparative amounts of Ser111-phosphorylated Rab1B and Rab8A and found that this disrupted the ability of Rab GTPases to interact with their effectors including cognate guanine exchange factor (GEF) Rabin8 and GTPase activating protein (GAP), TBCD1D4.

"Strikingly Katie also found that Ser111 phosphorylation disrupted the ability of LRRK2 to phosphorylate Threonine 72 on both Rab8A and Rab1B using in vitro phosphorylation assays. Together with post-docs Pawan Singh and Ilaria Volpi, they were able to establish transfection conditions in which Rab8A phosphorylation by PINK1 and LRRK2 could be observed in HEK293 cells. This revealed that Rab8A could indeed could undergo double phosphorylation by both PINK1 or LRRK2 at their respective sites but this was substantially less than expected compared to monophosphorylated forms of Rab8A suggesting antagonistic interplay between these phosphorylation sites in cells.

"During the course of her studies Katie and Yu-Chiang Lai undertook an in vitro screen for Rab kinases using the Dundee in-house panel of 150 recombinant kinases. Whilst they were unable to identify any kinases that could directly phosphorylate Rab8A at Ser111, they did discover that both MST3 and TAK1 could efficiently phosphorylate Rab8A at Thr72 in vitro in a similar manner to LRRK2. Interestingly Ser111-phosphorylated Rab8A does not impact on MST3 or TAK1-mediated Rab phosphorylation suggesting that SF3 Ser111 phosphorylation does not alter the accessibility of Thr72 on the Switch II domain.

"Consistent with this model, the Groll lab in TUM  were able to solve the x-ray crystal structure of Ser111-phosphorylated Rab8A and this did not reveal any major conformational changes in Rab8A compared to the dephosphorylated Rab8A structure. To assess whether there were any dynamic changes, the Sattler lab in TUM and Helmholz Centre in Munich obtained an NMR structure of Ser111-phosphoryated Rab1B and this also did not reveal any differences structurally from the dephosphorylated form.

"Overall these studies provide the first hints of cross-talk between the LRRK2 and PINK1 signalling pathways converging on the Rabs and suggests that Ser111 phosphorylation may impair the ability of LRRK2 to interact and bind to Rab8A. However, further work is required to validate this in physiological conditions in both cells and in vivo where PINK1 and LRRK2 are expressed at endogenous levels. The discovery that MST3 can efficiently phosphorylate Rab8A provides a facile method to obtain Thr72-phosphorylated Rab and this has already been exploited by the Alessi lab to study regulators and will be useful to the LRRK2 field at large."

The research was funded by the Michael J Fox Foundation, Parkinson’s UK, Wellcome Trust, Medical Research Council, and EMBO. The study involved collaborations with researchers at the Technical University of Munich, Helmholtz Zentrum München and the Universitätsklinikum Hamburg-Eppendorf (UKE)