Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle.

A cluster of phosphorylation sites in LRRK2 (leucine-rich repeat kinase 2), including Ser910, Ser935, Ser955 and Ser973, is important for PD (Parkinson's disease) pathogenesis as several PD-linked LRRK2 mutants are dephosphorylated at these sites. LRRK2 is also dephosphorylated in cells after pharmacological inhibition of its kinase activity, which is currently proposed as a strategy for disease-modifying PD therapy. Despite this importance of LRRK2 dephosphorylation in mutant LRRK2 pathological mechanism(s) and in LRRK2's response to inhibition, the mechanism by which this occurs is unknown. Therefore we aimed to identify the phosphatase for LRRK2. Using a panel of recombinant phosphatases, we found that PP1 (protein phosphatase 1) efficiently dephosphorylates LRRK2 in vitro. PP1 activity on LRRK2 dephosphorylation was confirmed in cells using PP1 inhibition to reverse LRRK2 dephosphorylation induced by the potent LRRK2 kinase inhibitor LRRK2-IN1 as well as in R1441G mutant LRRK2. We also found that PP1 and LRRK2 can form a complex in cells. Furthermore, we observed that PP1 inhibition modulates LRRK2's cellular phenotype by reducing skein-like LRRK2-positive structures associated with dephosphorylation. In conclusion, the present study reveals PP1 as the physiological LRRK2 phosphatase, responsible for LRRK2 dephosphorylation observed in PD mutant LRRK2 and after LRRK2 kinase inhibition.

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation.

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.

Generation of fourteen isogenic cell lines for Parkinson's disease-associated leucine-rich repeat kinase (LRRK2).

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with inherited forms of Parkinson's disease (PD), causing disease by a gain of kinase function. Here, we describe a series of isogenic iPSC lines with any of five pathogenic mutations (N1437H, R1441C, Y1699C, G2019S and I2020T); two hypothesis testing mutations (GTP binding null, T1348N, and kinase dead, K1906M) and two LRRK2 knockouts. This resource could be used to assess effects of mutations on the function of endogenous LRRK2 and/or to study LRRK2 interactors and substrates in iPSC-derived cellular models.