Fig 2: LRRK2 suppresses FAK Y397 phosphorylation through phosphorylation of TXR motif(s) in FAK.(a,b,d,f) In vitro kinase assays were carried out using 250 ng recombinant proteins of GST-FAK (a,d) FLAG-tagged WT, K454R mutant FAK (b) or T474A mutant FAK (f) and WT-, GS- and DA-LRRK2 recombinant proteins (50 ng in a,d or 50–200 ng in b and f) as indicated. 32P-labelled FAK was detected in autoradiograms (upper panels in a and b). Coomassie blue staining shows the amount of proteins in each reaction mixture (lower panels in a and b). In d and f, in vitro kinase assays were carried out using cold ATP without 32P-ATP, and the reaction mixtures were analysed by western blot using antibodies specific for FLAG, pTXR, FAK and LRRK2. Values are means±s.e.m. of three separate experiments. Data are representative of three independent experiments. (c) TXR(K) phosphorylation motifs found in FAK are indicated in bold. (e,g) Thr residues in six TXR sites (each number indicates the amino acid number in c) were mutated to Glu (T?E) (e). HEK 293T cells were transfected with FLAG-tagged WT-FAK or six (T?E) FAK mutants (e) or FLAG-tagged WT-FAK or T474A mutant FAK with GS-LRRK2 (g) for 48 h. Whole-cell lysates were prepared and immunoprecipitated with anti-FLAG antibody, and the levels of pY397-FAK and FLAG (e) or pTXR, Myc and FLAG (g) were analysed by western blot. One-way ANOVA with Newman–Keuls post hoc test in a and d *P<0.05, **P<0.01. Two-tailed Student's t-test in f. **P<0.01. ANOVA, analysis of variance.

Fig 3: LRRK2 negatively regulates FAK activation.(a) The effect of LRRK2 on FAK activation was analysed by western blot using antibodies specific for pY397-FAK, FAK and LRRK2 (upper panels) in LRRK2-KD cells (#1 and #2) and a control (NT) cells used in Fig. 1. Band intensities of pY397-FAK were quantified, normalized to that of FAK and plotted (lower panels). Values are means±s.e.m. of three separate experiments. (b) Microglia cultured from non-Tg or GS-Tg mouse brains were treated with 100 µM ADP for 5 min, and the levels of pY397-FAK, FAK and LRRK2 were analysed. Values are means±s.e.m. of five separate experiments. (c) Midbrain lysates were prepared from 8-week-old GS-Tg mice and littermate non-Tg mice. Each number indicates a different animal. Values are means ± s.e.m. of three or four animals. (d) HEK 293T cells were treated with ADP (100 µM) for the indicated times, and the levels of pY397-FAK were measured. (e) HEK 293T cells were transfected with Myc-tagged WT-LRRK2, GS-LRRK2 and DA-LRRK2 mutants. Forty-eight hours later, cells were treated with ADP (100 µM) for the indicated times. Data are representative of three independent experiments. Values are means±s.e.m. of three independent experiments. One-way ANOVA with Newman–Keuls post hoc test, *P<0.05, **P<0.01 in a,b and e. Two-tailed Student's t-test, **P<0.01 in c. ANOVA, analysis of variance.

Fig 4: The LRRK2 kinase inhibitor, GSK2578215A (GSK), reduces pTXR-FAK levels and rescues pY397-FAK levels and motility of GS-Tg microglia.(a) HEK 293T cells expressing GS-LRRK2 were treated with ADP (100 µM) for 5 min in the presence of the indicated amount of GSK. Levels of S935-autophosphorylated LRRK2 (pS935-LRRK2) and Y397-autophosphorylated FAK (pY397-FAK) were measured by western blot using antibodies specific for pS935-LRRK2 and pY397-FAK, respectively. FAK and Myc were used as loading controls. Band intensities were quantified and plotted. Values are means±s.e.m. of three separate experiments. (b) In vitro kinase assays were carried out using recombinant GST-FAK and GS-LRRK2 (GS) or DA-LRRK2 (DA) in the absence or presence of the indicated amount of GSK. pTXR-FAK was analysed and plotted. Values are means±s.e.m. of three separate experiments. (c–e) GS-Tg microglia were treated with GSK (1 µM) for 30 min and then treated with ADP. ADP (100 µM) induced formation of stable lamellipodia followed by cell body movement in the presence of GSK (c, arrowheads). SACED showed that GSK (n=26) increased protrusion compared to DMSO (n=32) (d,e). One-way ANOVA with Newman–Keuls post hoc test, *P<0.05, **P<0.01, ##P<0.01 in a and b. Two-tailed Student's t-test, **P<0.01 in e. Scale bar, 50 µm (c). ANOVA, analysis of variance.

Fig 5: Modeling of full-length LRRK2 into the cryo-ET reconstruction of microtubule-associated LRRK2[I2020T] filaments in cells.a, Cryo-ET reconstruction of microtubule-associated LRRK2[I2020T] filaments in cells1. The LRRK2 strands that form the double-helical filaments are shown in light and dark orange. For this figure, the density corresponding to the microtubule was replaced with a 10 Å representation of a molecular model of a microtubule. b, We docked copies of our 5.9 Å reconstruction of a LRRK2RCKW[I2020T] tetramer from the microtubule-associated filaments (Fig. 1b, c) into the regions indicated by the parallelograms in (a). c, Next, we docked two copies of the AlphaFold model of full-length LRRK2 (AF-Q5S007), which is in the active state, as is the case with LRRK2RCKW[I2020T] in our filaments, into each of the 5.9 Å maps. The pairs of AlphaFold models in each map correspond to the COR-B:COR-B dimer. This panel shows a region corresponding to the rectangle in (b). d, Three different views of the models docked in (c). Below each model, close-ups show regions where adjacent filaments clash. These clashes involve a domain in the N-terminal repeats of one LRRK2, and either the same domain on another LRRK2, or the WD40 domain. For clarity, one of the LRRK2’s is shown in grey instead of the standard rainbow coloring.