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Rab GTPases as Physiological Substrates of LRRK2 Kinase.

ABSTRACT: LRRK2 (Leucine-Rich Repeat Kinase 2) is a gene whose specific mutations cause Parkinson's disease (PD), the most common neurodegenerative movement disorder. LRRK2 harbors GTPase and kinase activities, two enzyme activities that play critical roles in the regulation of cellular signal transduction. Among the several LRRK2 pathogenic mutations, the most prevalent G2019S mutation increases its kinase activity when compared with the wild-type (WT), suggesting that LRRK2 kinase substrates are potential culprits of PD pathogenesis. Although there were several studies to identify LRRK2 kinase substrates, most of them mainly employed in vitro kinase assays. Therefore, it remains uncertain whether the identified substrates were real physiological substrates. However, efforts to determine physiological LRRK2 kinase substrates have recently identified several members of the Rab GTPase family as physiological LRRK2 kinase substrates. A conserved threonine or serine in the switch II domain of certain Rab GTPase family members (Rab3A/B/C/D, Rab5A/B, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) has been pinpointed to be phosphorylated by LRRK2 in cells using sophisticated phosphoproteomics technology in combination with LRRK2-specific kinase inhibitors. The Rab GTPases regulate vesicle trafficking, suggesting that LRRK2 may be a regulator of such vesicle trafficking, confirming previously suggested LRRK2 functions. However, how the consequence of the LRRK2-mediated Rab phosphorylation is related to PD pathogenesis is not clear. This review briefly summarizes the recent results about LRRK2-mediated Rab phosphorylation studies.

SUBMITTER: Seol W

PROVIDER: S-EPMC6526114 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Rab GTPases as Physiological Substrates of LRRK2 Kinase.

Seol Wongi W Nam Daleum D Son Ilhong I

Experimental neurobiology 20190430 2

LRRK2 (Leucine-Rich Repeat Kinase 2) is a gene whose specific mutations cause Parkinson's disease (PD), the most common neurodegenerative movement disorder. LRRK2 harbors GTPase and kinase activities, two enzyme activities that play critical roles in the regulation of cellular signal transduction. Among the several LRRK2 pathogenic mutations, the most prevalent G2019S mutation increases its kinase activity when compared with the wild-type (WT), suggesting that LRRK2 kinase substrates are potenti ...[more]

PMID: 31138985

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Project description:BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.MethodsIn vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.ResultsOur screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.ConclusionsHere we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

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Rab GTPases as Physiological Substrates of LRRK2 Kinase.

Publications

Rab GTPases as Physiological Substrates of LRRK2 Kinase.

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