Figure 1. GTP is a phospho-donor for LRRK2 kinase activity.

A) Schematic of the human GST-[Δ970]LRRK2 and isolated ROC domain with annotation relative to the full length LRRK2 protein (NCBI entry NP_940980.3), and a LRRK2 small-peptide kinase substrate. B) WT, G2019S (kinase over-active) and D1994A (kinase-dead) LRRK2 incubated with 1 mM GTP and 1.7 nM [γ-32P]-GTP in the presence of 10 mM MnCl₂. Coomassie staining showed the purity of the recombinant proteins. ³²P incorporation is demonstrated with autoradiography. C) WT, G2019S and D1994A recombinant LRRK2 proteins were incubated in kinase assays with 1 mM (cold) ATP or GTP in the presence of 10 mM Mg²⁺ or Mn²⁺, as indicated. LRRK2 autophosphorylation was measured by a phospho-threonine specific antibody from 30 min kinase reactions. A representative immunoblot is shown. Total recombinant protein in the reaction was measured using a GST antibody. D) The absolute amount of Pi incorporated during LRRK2-autophosphorylation was calculated by measuring by [γ-³²P] ATP (top panels) or GTP (bottom panels, as indicated) incorporation into LRRK2 protein (G2019S, gray bars, WT, white bars) in the presence of 1 mM ATP or GTP (cold) with 1.7 nM [γ-³²P] ATP or [γ-³²P] GTP and 10 mM MnCl₂ or MgCl₂ (as indicated), followed by liquid scintillation and normalization to standard curves of Pi that correct for efficiency in detection. Of note, all autophosphorylation signal in reactions with GTP and Mg²⁺ were not detectable above baseline (background). E) LRRK2 trans phosphorylation of a small peptide substrate (1 nmole per reaction) was measured in reactions supplemented with 1.7 nM [γ-³²P] ATP or GTP with 1 mM (cold) ATP or GTP. F) G2019S-LRRK2 incubated with peptide substrate and 1 mM GTP supplemented with 1.7nM [γ-³²P] GTP in the presence of 1 μM of the indicated ATP-competitive LRRK2 kinase inhibitor, or DMSO control (0.1% of reaction volume), with kinase dead (D1994A) LRRK2 that fails to produce significant peptide phosphorylation. All graphs depict the mean value from three independent experiments with S.E.M error bars. * p<0.05, two-way ANOVA with Tukey’s post-hoc test.