LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

Abstract

Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations

“…The most prevalent PD mutation is G2019S in the kinase domain, which enhances kinase activity, whereas the PD-related mutation I2020T shows slightly decreased activity (15,16,(28)(29)(30). The LRRK2 G2019 and I2020 residues are conserved in Roco4 and correspond to G1179 and L1180, respectively (Fig.…”

Update on the Functional Biology of Lrrk2

“…Since then, the G2019S mutation has been reported to enhance kinase activity compared with WT activity [45,47,55,56]. The I2020T mutation has been reported to increase kinase activity [44,57] but also to reduce kinase activity [55]. Data on kinase alterations by mutation at position R1441 are conflicting, with reports of no change [20,55,58] and increased [47,57,59] for R1441C; and unaltered [55] and increased [57] for R1441G.…”

“…The I2020T mutation has been reported to increase kinase activity [44,57] but also to reduce kinase activity [55]. Data on kinase alterations by mutation at position R1441 are conflicting, with reports of no change [20,55,58] and increased [47,57,59] for R1441C; and unaltered [55] and increased [57] for R1441G. Finally, the Y1699C mutation is reported as unaltered [20,55] and increased [57].…”

“…Since then, the G2019S mutation has been reported to enhance kinase activity compared with WT activity [45,47,55,56]. The I2020T mutation has been reported to increase kinase activity [44,57] but also to reduce kinase activity [55].…”

“…Kinase substrate tracking and elucidation (KESTREL) screening has shown that Lrrk2 may phosphorylate three closely related proteins, ezrin, radixin and moesin (known as ERM proteins), at their analogous residues Thr 567, Thr 564 and Thr 558 [55]. Moesin and radixin are involved in neurite growth cone morphology, motility and process formation in primary cultured neurons [65].…”