Abstract
Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A "substrate-trapping" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.
Highlights
LRRK2 is a large multidomain protein enzyme consisting of a Roc-Cor family GTPase followed by a serine/threonine kinase domain (Cookson, 2010; Gilsbach and Kortholt, 2014)
We first explored whether PP1 (Protein Phosphatase-1) or PP2A (Protein Phosphatase-2A) previously implicated in regulating LRRK2 (Lobbestael et al, 2013; Athanasopoulos et al, 2016), control Rab phosphorylation
Wild-type and LRRK2[R1441C] knock-in mouse embryonic fibroblasts (MEF cells) were treated with 0.1 mM calyculin-A (PP1 inhibitor) or 1 mM Okadaic acid (PP2A inhibitor) (Ishihara et al, 1989), and neither of these agents had a significant impact on basal phosphorylation of Rab10 (Thr73) or Rab12 (Ser105) or dephosphorylation following administration of the well-characterized highly selective and potent LRRK2 inhibitor termed MLi-2 (Fell et al, 2015) (Figure 1)
Summary
LRRK2 is a large multidomain protein enzyme consisting of a Roc-Cor family GTPase followed by a serine/threonine kinase domain (Cookson, 2010; Gilsbach and Kortholt, 2014). Missense mutations within the GTPase domain (N1437H, R1441G/C, Y1699C) and kinase domains (G2019S, I2020T) hyper-activate LRRK2 protein kinase and cause Parkinson’s disease (PD) (Zimprich et al, 2004; Paisan-Ruız et al, 2004; Alessi and Sammler, 2018). Mutations in LRRK2 are one of the most common genetic causes of familial Parkinson’s comprising ~5% of familial Parkinson’s, and ~1% of sporadic Parkinson’s patients (Healy et al, 2008; Domingo and Klein, 2018). In terms of clinical presentation and late age of onset, LRRK2 mediated Parkinson’s closely resembles the common sporadic form of the disease affecting the vast majority of patients (Marras et al, 2011). Pharmaceutical companies have developed LRRK2 inhibitors for treatment and prevention of PD (Alessi and Sammler, 2018) and clinical trials have commenced and/or are planned (see https://clinicaltrials.gov/ NCT03976349 (BIIB094) and NCT03710707 (DNL201))
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