Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease and activated LRRK2 phosphorylates a subset of Rab GTPases. Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this site, termed “Site #1”, can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed “Site #2”, that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments and mutation analysis demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab reaction products. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and apparent membrane activation of LRRK2 kinase activity.

Figure 1 zip file:

-Figure1ABCD_ Prism files containing tabular data of mst experiments for recombinant Rab29 binding to LRRK2 Armadillo 1-552,1-159, 350-550, and Rab7 binding to LRRK2 Armadillo 1-552.

Figure 2 zip file:

-Figure2ABC_ Prism files containing tabular data of mst experiments for recombinant Rab8A binding to LRRK2 Armadillo 1-552,1-159, and 350-550.

-Figure2DEF_ Prism files containing tabular data of mst experiments for recombinant Rab10 binding to LRRK2 Armadillo 1-552,1-159, and 350-550.

Figure3 zip file:

-Figure3B_ Prism file containing tabular data of GFP LRRK2 WT and HA Rab29 colocalization as measured by Mander's coefficient.

-Figure3S1_ Images of GFP LRRK2 Nterminal truncations amd HA Rab29 co-expressing HeLa cells.

-Figure3S3_ Images of GFP LRRK2 WT and GFP LRRK2 point mutants and HA Rab29 co-expressing HeLa cells used to generate figureB.

-Figure3S4_ PDF of annotated 700 and 800 channel for immunoblots used to generate figure3C and figure3D.

Figure4 zip file:

-Figure4ABC_ Prism files containing tabular data of mst experiments for recombinant phosphoRab8A binding to LRRK2 Armadillo 1-552,1-159, and 350-550.

-Figure4DEF_Prism files containing tabular data of mst experiments for recombinant phosphoRab10 binding to LRRK2 Armadillo 1-552,1-159, and 350-550.

Figure5 zip file:

-Figure5A_ Electrostatic mapping of LRRK2 Armadillo model.

-Figure5CD_ Prism files containing tabular data of mst experiments for recombinant phosphoRab10 binding to LRRK2 Armadillo 1-552 K17A or K18A.

Figure6 zip file:

-Figure6AB_ Images of FLAG LRRK2 R1441G and FLAG LRRK2 R1441GK17AK18A expressing HeLa cells. Images from three different experiment per condition.

-Figure6C_ Prism files containing tabular data of quantification by Mander’s Coefficient colocalization analysis.

Figure7 zip file:

-Figure7RAWdata_ 700 and 800 channels of blots for figure7ABCD.

-Figure7EF_ Prism files containing tabular data of quantification of blots of MLi2 washout experiments.

Figure8 zip file:

-Figure8RAWdata_ TIRF movies of LRRK2 recruitment to Rab10-lipid bilayers.

-Figure8A_ Prism files containing tabular data of quantification of R1441G, D2017A, or R1441GK17AK18A LRRK2 recruitment to Rab10-lipid bilayers fitted to a non-linear regression curve.

-Figure8B_ Prism files containing tabular data of quantification of R1441G LRRK2 recruitment to noRab10 or Rab11-lipid bilayers fitted to a non-linear regression curve.

-Figure8C_ Prism files containing tabular data of quantification of 0.18-2.5uM R1441G LRRK2 recruitment to Rab10-lipid bilayers fitted to a non-linear regression curve. Initial rates then plotted in separate prism file and fitted to a nonlinear regression curve to determine a Hill coefficient.

Figure8S2_ 700 and 800 channels of blots for figure for figure 8S2.

Figure9 zip file:

-Figure9RAWdata_ 700 and 800 channels of blots for figure9AB.

-Figure9CD_ Prism files containing tabular data of quantification of blots in figure9AB.