ArfGAP1 enhances the GTP hydrolysis activity of LRRK2.

<p>(A) ArfGAP1-YFP expression fails to influence the steady-state levels of FLAG-tagged WT LRRK2 bound to GTP following pull-down assays with GTP-sepharose from HEK-293T cells. (B) Confirmation of the specificity of LRRK2 GTP binding is indicated by negligible binding of the GDP/GTP binding-deficient LRRK2 mutant, T1348N. Competition with an excess of free GTP (2 mM) abolishes binding of WT LRRK2 to GTP-sepharose in the presence or absence of ArfGAP1. Each input lysate (<i>top panel</i>) was split equally and used for GTP pull-down assays in the presence or absence of free GTP (<i>lower panel</i>), as indicated. (C) ArfGAP1-YFP expression fails to influence the steady-state levels of GTP-bound WT and PD-associated mutant LRRK2. Densitometric analysis reveals a significant increase in GTP binding of the R1441C and Y1699C LRRK2 variants compared to WT LRRK2, independent of ArfGAP1 expression. Data represent the level of LRRK2 GTP binding expressed as a percent of WT LRRK2 levels. GTP-bound LRRK2 levels were normalized to LRRK2 input levels. Bars represent the mean ± SEM (<i>n</i> = 3 experiments). *<i>P</i><0.05 and **<i>P</i><0.01 compared to WT LRRK2 by one-way ANOVA with Newman-Keuls post-hoc analysis. (D) GTP hydrolysis activity of Arf1 in the presence or absence of full-length ArfGAP1 was determined by measuring the concentration of free phosphate (P_i) released from GTP. WT ArfGAP1 enhances Arf1-mediated GTP hydrolysis whereas ΔN ArfGAP1 has negligible effects on Arf1. The input levels of immunopurified Arf1-CFP and ArfGAP1-YFP variant were assessed by Western blot analysis with anti-GFP antibody (<i>lower panel</i>). (E) GTP hydrolysis activity of WT LRRK2 is significantly enhanced by WT ArfGAP1 but not by ΔN ArfGAP1. Guanine nucleotide-deficient T1348N LRRK2 exhibits impaired GTP hydrolysis activity, whereas the GTPase-hyperactive R1398L LRRK2 exhibits enhanced activity compared to WT LRRK2. The input levels of immunopurified FLAG-LRRK2 variants were assessed by Western blot analysis with anti-FLAG antibody (E, <i>upper panel</i>), quantified by densitometry, and used for normalization of P_i release between LRRK2 variants. GTP hydrolysis activity for Arf1 (D) or LRRK2 (E) is expressed as P_i release as a percent of Arf1 or WT LRRK2 activity alone, as indicated. Bars represent the mean ± SEM (<i>n</i> = 5 experiments). *<i>P</i><0.05 and **<i>P</i><0.01 compared to Arf1 (D) or WT LRRK2 (E) by one-way ANOVA with Newman-Keuls post-hoc analysis.