Abstract
BackgroundSeveral mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson’s disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth.MethodsThe phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot.ResultsOur assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin.ConclusionsY1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.
Highlights
Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson’s disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability
Rab8a is phosphorylated by LRRK2 In order to confirm whether Rab8a could be phosphorylated by LRRK2, we overexpressed FLAG-tagged Rab8a
The T72A mutant of Rab8a could not be phosphorylated by LRRK2 (Y1699C), indicating that the phosphorylation of Rab8a by LRRK2 Y1699C exclusively occurs on T72 site
Summary
Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson’s disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Rabs are a group of about 70 eukaryotic proteins located on different membranes that play important roles in all stages of vesicle trafficking including budding, motility and fusion [1, 2]. The most important feature of Rab proteins is that they can switch from inactive GDP-bound form to active GTP-bound form and recruit unique effectors which play their roles in vesicle trafficking [3]. The. Rab has shown to play important roles in membrane trafficking. Rab has shown to play important roles in membrane trafficking It plays irreplaceable roles in multiple biological processes including cellular morphology, cell polarity, cell movement, neural differentiation and ciliogenesis [8].
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