LRRK2-mediated regulation of association/dissociation between tau and tubulin.

<p>LRRK2 interacts with tubulin-associated tau, resulting in the formation of a tripartite complex (lower left). This complex induces the phosphorylation of tau by LRRK2 (upper left) and sequentially induces dissociation of tau from tubulin (upper right). Tau is dephosphorylated by certain protein phosphatases, and the dephosphorylated tau then recovers its ability to bind with tubulin (lower right).</p

Tubulin-dependent interaction of LRRK2 with tau.

<p>(A) V5-LRRK2 was immunoprecipitated with anti-V5-agarose beads from the V5-LRRK2-stably-expressing SH-SY5Y clone, WT4-D33. Normal rabbit IgG-conjugated agarose beads were used as a control. The immunoprecipitates were separated by SDS-PAGE, transferred to PVDF, and analyzed using antibodies against V5-tag, tau, and tubulin. (B) GST (lane 1) and GST-LRRK2 (lane 2) were incubated with porcine tubulin, and a pull-down assay using glutathione-agarose beads was performed. Precipitated proteins were detected by Western analysis using anti-GST and anti-tubulin antibodies. (C) GST and GST-LRRK2 were incubated with recombinant tau in the absence (lane 1 and 2) or presence (lane 3 and 4) of porcine tubulin, and a pull-down assay using glutathione-agarose beads was performed. Precipitated proteins were detected by Western analysis using the indicated antibodies.</p

Effect of LRRK2-mediated phosphorylation on the ability of tau to bind to tubulin.

<p>(A) Recombinant tau (2 µg) was incubated with GST-LRRK2 (50 ng) and 3 µCi of [γ-³²P]ATP in the presence of porcine tubulin (1 µg) at 30°C for 120 min and then a GST pull-down assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030834#s2" target="_blank">Materials and Methods</a>. ³²P-Labeled proteins in the precipitates were detected by autoradiography following SDS-PAGE (upper panel). Total tau in the same sample was detected by Western blotting analysis using anti-tau antibody (lower panel). (B) V5-LRRK2 was immunoprecipitated with anti-V5-agarose beads from a WT4-D33 cell lysate. Precipitated proteins were analyzed by Western blotting using antibodies against V5-tag, phospho-tau (Thr181), non-phospho-epitopes of tau, and tubulin.</p

Effect of overexpression and knockdown of LRRK2 on phosphorylation of tau (Thr181) in neuronal cells and their neurite outgrowth.

<p>(A) Left: A SH-SY5Y clone (WT4-D33) expressing V5-tagged wild-type LRRK2 was transfected with either the LRRK2-specific RNAi or the RNAi control. After 48 h of transfection, cell lysates were prepared and subjected to Western analysis using antibodies against V5-tag, LRRK2, phospho-tau (Thr181), and non-phosphorylated tau. A vector control clone (NEO) expressing only the neomycin gene is shown in lane 1. Right: Graphical representation of the tau (Thr181)-phosphorylation level. (B) Left: WT4-D33 and NEO were treated with 10 µM all-trans retinoic acid for 72 h, transfected with the LRRK2-specific RNAi or RNAi-control, cultured for a further 72 h, and subjected to immunostaining with anti-β III tubulin. Right: Graphical representation of neurite length measured using NIH ImageJ software. Stars represent statistical comparisons by one-way ANOVA (n = 3 in A and n = 50 in B); *: p<0.005, **: p<0.001.</p

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

<p>(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.</p