Fig. 5.

GTPase activity in Leucine-rich repeat kinase 2 (LRRK2) GTPase domain and decreased GTP hydrolysis in GTPase domain containing pathogenic Parkinson’s disease (PD) mutation R1441C/G. (a) A diagram shows schematic of the full-length of LRRK2 protein containing functional domains, wild type GTPase domain (from aa1321 to aa1516), mutations in GTPase domain R1441C or G and T1348N. (b) FLAG-GTPase domain of wild type and mutant variants as indicated were purified from HEK-293T cells after transfection, followed by analysis on SDS-PAGE and visualization by Coomassie blue staining and western blot (WB) detection with anti-FLAG antibody. The protein levels were normalized for the GTP hydrolysis assay. (c) GTP hydrolysis of purified GTPase domain of wild type and mutant variants was assayed by measurement of the release of γ-³²P from [γ-³²P]-GTP at 40 min. The value of wild type GTPase domain is significantly higher than mutants R1441C (p < 0.05, student’s t test), R1441G (p < 0.05, student’s t test), and T1348N (p < 0.0001, student’s t test). The result is presented as mean values from three independent experiments (±SEM). (d) GTPase domain directly binds to GTP. The cell lysates (100 μg) from transfected cells with plasmids expressing GTPase domain of wild type, or individual mutant variant was incubated with GTP sepharose. The precipitated products by GTP sepharose pull-down were resolved by SDS-PAGE and detected by immunoblot analysis with anti-FLAG antibody. The presence of 2 mmol/L GTP but not ATP eliminates the binding of wild type GTPase domain to GTP sepharose. Wild type GTPase domain shares similar GTP binding efficiency to GTPase domain mutants R1441C or R1441G. (e) PD mutant R1441C/G show no significant difference (p = 0.2, student’s t test) in GTPγS binding as compared to wild type GTPase domain. The GTPγS loading of purified wild type GTPase domain and PD mutant R1441C/G was measured by filter-binding assay and result is presented as mean value (±SEM) from three independent experiments.