Abstract
Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.
Highlights
Parkinson’s disease (PD) is a progressive neurodegenerative disease affecting 1–2% of the population over 65 years of age, with approximately 60,000 newly diagnosed patients per year
We first wished to test the in vitro utility of the autophosphorylation site antibodies as indicators of Leucine-Rich Repeat protein Kinase 2 (LRRK2) kinase activity in an isolated system, to which we could compare the effects we observe in cellular studies
The PD causing mutations Gly2019Ser and Arg1441Cys exhibited enhanced Ser1292 phosphorylation compared to wild-type, and kinase inactive LRRK2 is not modified at Ser1292 whereas the cellular sites are, Figure 1B
Summary
Parkinson’s disease (PD) is a progressive neurodegenerative disease affecting 1–2% of the population over 65 years of age, with approximately 60,000 newly diagnosed patients per year. The increasing disability caused by the progression of disease burdens the patients, their caregivers, as well as society. Hallmark clinical features of PD include resting tremor, bradykinesia, postural instability and rigidity. PD exhibits a wide variety of non-motor features such as autonomic dysfunction and dementia. The pattern of neuronal loss in PD is well-characterized, the molecular mechanisms of progressive cell death are still being elucidated. Exposure to a number of environmental toxicants has been shown to increase the risk of PD. Understanding the mechanisms of these known causes and risks will likely lead to the development of novel therapeutics, an unmet need
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