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

Mahaboobi Jaleel , R Jeremy Nichols , Maria Deak , David G Campbell , Frank Gillardon , Axel Knebel , Dario R Alessi (1)

1 College of Life Sciences

Mutations in the Leucine Rich Repeat Kinase-2 (LRRK2) gene cause late-onset Parkinson's disease (PD). LRRK2, contains Leucine Rich Repeats, a GTPase domain, a COR domain, a kinase and a WD40 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 myelin basic protein, which is catalysed rather slowly. We undertook a KESTREL 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 that are related to moesin, at the residue equivalent to Thr558, as well as a peptide encompassing Thr558 (LRRKtide). 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 not only caused by an increase of 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 WD40 motif and C-terminal tail. This study suggests that moesin, ezrin and radixin may comprise LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.

Mots clés: Life Sciences

Mahaboobi Jaleel, R Jeremy Nichols, Maria Deak, David G Campbell, Frank Gillardon, et al.. LRRK2 phosphorylates moesin at Threonine-558: characterization of how Parkinson‘s disease mutants affect kinase activity. Biochemical Journal, 2007, 405 (2), pp.307-318. ⟨10.1042/BJ20070209⟩. ⟨hal-00478755⟩