The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Abstract

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate the how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…Various PDassociated mutations in LRRK2 have been shown to augment kinase activity (6,7,11,12) and increase the phosphorylation of Rab proteins, recently identified as likely physiological substrates (12). In addition, LRRK2 mutations within the Roc domain have been associated with decreased LRRK2 GTPase activity (13,14).…”

Update on the Functional Biology of Lrrk2

“…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].…”

“…Several groups now have shown that Lrrk2 can bind GTP via GTP-sepharose or GTPγS-binding assays, whereas disruption of the P-loop in the nucleotide-binding pocket of the Roc domain abolishes GTP binding [33,47,53,[57][58][59][60]. GTPase activity has been more difficult to demonstrate [57,60], presumably owing to the requirement of GEFs and GAPs for in vitro assays; however, optimized experimental conditions have now enabled GTPase activity, albeit low, to be detected [33,53,58,59].…”

“…GTPase activity has been more difficult to demonstrate [57,60], presumably owing to the requirement of GEFs and GAPs for in vitro assays; however, optimized experimental conditions have now enabled GTPase activity, albeit low, to be detected [33,53,58,59]. Several groups now agree that Lrrk2 Roc domain mutations do not increase GTP binding [33,58,59], but Roc mutants do reduce GTPase activity [33,58,59] and suggest that Roc domain mutations prolong Lrrk2 in the active GTP-bound state. In support of this, crystal structure studies show that the active GTPase domain of Lrrk2 has a unique dimeric fold and support a prolonged hydrolysis hypothesis, the prediction being that mutations in the ROC domain disrupt this tertiary structure [53].…”

“…Indeed, Lrrk2-mediated phosphorylation is stimulated by binding of nonhydrolyzable GTP analogs, suggesting that Lrrk2 is activated by MAPKKK intramolecularly by its own GTPase [59]. Furthermore, Roc domain mutations R1441C/G lead to enhanced kinase activity, presumably by prolonging Lrrk2 in the active GTP-bound state [57,59] (although one report did not replicate this finding [58]). It remains to be seen whether GTPase activity can regulate kinase activity in vivo, for which the development of animal models will be imperative, and to subsequently test the therapeutic benefit of targeting GTPase activity.…”