Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant

Abstract

J. Neurochem. (2011) 116, 304–315. Abstract Mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson’s disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C‐terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC‐COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra‐molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra‐molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC‐COR tandem of LRRK2, showing that Y1699 is positioned at the intra‐molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra‐molecular ROC : COR interface, strengthens the intra‐molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC‐COR tandem domain resulting in decreased GTPase activity.

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

“…In agreement with this model, PKA phosphorylation and subsequent binding of 14-3-3 might regulate LRRK2 kinase activity negatively by preventing dimerization of its ROC domain. Considering the recent findings by Daniëls et al (45), this negative regulation also might be mediated by 14-3-3, affecting the interaction of ROC with the adjacent COR domain. Future studies are needed to correlate PKA-mediated phosphorylation of pS1444 with 14-3-3 binding and LRRK2 activity in vivo.…”

Heterogeneity of Leucine-Rich Repeat Kinase 2 Mutations: Genetics, Mechanisms and Therapeutic Implications

“…The R1441 mutations cause a decrease in GTPase activity of LRRK2, although this activity is low and has been difficult to measure reliably, leading to some uncertainty about relevance of this observation [18][19][20]88]. The mechanism of diminished activity is likely via disruption of hydrogen bonding and stacking interactions normally provided by the arginine side chain [18][19][20]87], which may cause an enhancement in the affinity of LRRK2 for GTP with a subsequent decrease in GTP hydrolysis [89].…”

“…The Y1699 residue is located on the outer surface of the COR domain and substitution with cytosine at this likely alters interactions between the ROC and COR domains, leading to a reduction in GTPase activity [88,91]. As for R1441 mutations, the kinase function of Y1699C LRRK2 is similar to wild-type protein [13,44,67,68].…”

Assaying the Kinase Activity of LRRK2 <em>in vitro</em>

“…Recently, Daniëls et al reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available 13 . In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity.…”

“…Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK2 12 . Recently, Daniëls et al reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available 13 . In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity.…”