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Updated: Jul 23, 2025
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
LRRK2 phosphorylation status and kinase activity regulate (macro)autophagy in a Rab8a/Rab10-dependent manner
Elżbieta Kania1,2,3, Jaclyn S Long1,2, David G McEwan1,2
1Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
Abstract:
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.
Insights
Leucine-rich repeat kinase 2 (LRRK2) phosphorylation sites regulate autophagy. Impaired LRRK2 phosphorylation disrupts cellular waste removal, contributing to Parkinson's disease pathology by increasing kinase activity.
Area of Science:
- Cellular Biology
- Molecular Biology
- Neuroscience
Background:
- Mutations in leucine-rich repeat kinase 2 (LRRK2) are a leading genetic cause of Parkinson's disease (PD).
- LRRK2 protein possesses GTPase and kinase activity, influenced by its phosphorylation status.
- The role of constitutive phosphorylation sites (S910/S935/S955/S973) in LRRK2 function is largely unknown.
Purpose of the Study:
- To investigate the role of LRRK2 phosphorylation at S910/S935/S955/S973 sites in regulating autophagy.
- To determine the impact of altered LRRK2 phosphorylation on cellular lysosomal function and autophagic flux.
- To elucidate the mechanistic link between LRRK2 kinase activity, phosphorylation status, and autophagy impairment in Parkinson's disease models.
Main Methods:
- Generated quadruple LRRK2 phosphomutant cells (4xSA) to mimic reduced phosphorylation.
- Utilized LRRK2 kinase inhibitors (MLi-2, PF-06447475) to assess the effect of kinase activity modulation on autophagy.
- Analyzed downstream LRRK2 phosphorylation targets (Rab8a, Rab10) and their role in autophagy.
- Examined cells expressing the pathogenic LRRK2 R1441C mutant.
Main Results:
- Cells with quadruple LRRK2 phosphomutant (4xSA) exhibited impaired lysosomal function and failed to induce autophagy during starvation.
- LRRK2 kinase inhibitors, despite reducing phosphorylation at S910/S935/S955/S973, did not affect autophagy.
- Autophagy impairment in 4xSA cells was attributed to enhanced LRRK2 kinase activity, evidenced by increased phosphorylation of Rab8a and Rab10.
- Expression of phosphorylation-deficient Rab8a and Rab10 mutants rescued the autophagy defect.
- Pathological LRRK2 R1441C mutant cells showed reduced autophagy and decreased constitutive phosphorylation, correlating with increased kinase activity.
Conclusions:
- LRRK2 phosphorylation at S910/S935/S955/S973 sites is critical for regulating autophagy under basal and starvation conditions.
- Enhanced LRRK2 kinase activity, rather than direct phosphorylation status at these sites, drives autophagy impairment in LRRK2-associated Parkinson's disease.
- These findings highlight the intricate relationship between LRRK2 phosphorylation, kinase activity, and autophagy, offering insights into Parkinson's disease pathogenesis.
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