Figure 2. LRRK2-mediated phosphorylation of tau in the presence of tubulin.

(A) Recombinant tau (2 µg) was incubated with GST-LRRK2 (50 ng each) and 3 µCi of [γ-³²P]ATP in the absence (lane 1) or presence (lane 2) of purified porcine tubulin (1 µg) for 30 min at 30°C. ³²P-Labeled proteins in the reaction mixture were detected by autoradiography following SDS-PAGE. A Coomassie Brilliant Blue (CBB) post-stained gel image is shown in the right panel to indicate the identity and amount of LRRK2 and tau in the two lanes. (B) The kinetics of tau phosphorylation by LRRK2 was measured after incubation for indicated periods at 30°C. The radioactivity of ³²P-labeled tau in the presence (•) or absence (○) of tubulin was determined using a liquid scintillation counter. (C) Equal amounts (2 µg) of tau, tubulin, and MBP were incubated with GST-LRRK2 (50 ng each) and 3 µCi of [γ-³²P]ATP for 30 min at 30°C. The radioactivity of ³²P-labeled proteins was determined using a liquid scintillation counter. (D) Left: Tau (2 µg) was incubated with GST-LRRK2 of the wild-type (WT), G2019S, I2020T, or R1441C mutants and 3 µCi of [γ-³²P]ATP in the presence of tubulin (1 µg) for 30 min at 30°C. The ³²P-labeled proteins in the reaction mixture were detected by autoradiography following SDS-PAGE. CBB post-stained gel images are shown in the bottom panel to indicate the identity and amount of LRRK2, tau, and tubulin in the lanes. Right: Graphical representation of the rates of tau phosphorylation by WT, G2019S, I2020T, and R1441C LRRK2. Phosphorylation rates relative to WT-LRRK2 are shown.