DOI: 10.5061/dryad.3tx95x6j7
title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and apparent activation

description: 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.
description: <strong>Figure 1 zip file:</strong>
 <strong>-Figure1ABCD</strong>_ 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. <strong>Figure 2 zip
 file:</strong>
 <strong>-Figure2ABC</strong>_ Prism files containing
 tabular data of mst experiments for recombinant Rab8A binding to LRRK2
 Armadillo 1-552,1-159, and 350-550.
 <strong>-Figure2DEF</strong>_ Prism files containing
 tabular data of mst experiments for recombinant Rab10 binding to LRRK2
 Armadillo 1-552,1-159, and 350-550.
 <strong>Figure3 zip file:</strong>
 <strong>-Figure3B</strong>_ Prism file containing
 tabular data of GFP LRRK2 WT and HA Rab29 colocalization as measured by
 Mander's coefficient.
 <strong>-Figure3S1</strong>_ Images of GFP LRRK2
 Nterminal truncations amd HA Rab29 co-expressing HeLa cells.
 <strong>-Figure3S3</strong>_ Images of GFP LRRK2 WT
 and GFP LRRK2 point mutants and HA Rab29 co-expressing HeLa cells used to
 generate figureB.
 <strong>-Figure3S4</strong>_ PDF of annotated 700 and
 800 channel for immunoblots used to generate figure3C and
 figure3D. <strong>Figure4 zip
 file:</strong>
 <strong>-Figure4ABC</strong>_ Prism files containing
 tabular data of mst experiments for recombinant phosphoRab8A binding to
 LRRK2 Armadillo 1-552,1-159, and 350-550.
 <strong>-Figure4DEF</strong>_Prism files containing
 tabular data of mst experiments for recombinant phosphoRab10 binding to
 LRRK2 Armadillo 1-552,1-159, and 350-550.
 <strong>Figure5 zip file:</strong>
 <strong>-Figure5A</strong>_ Electrostatic mapping of
 LRRK2 Armadillo model.
 <strong>-Figure5CD</strong>_ Prism files containing
 tabular data of mst experiments for recombinant phosphoRab10 binding to
 LRRK2 Armadillo 1-552 K17A or K18A.
 <strong>Figure6 zip file:</strong>
 <strong>-Figure6AB</strong>_ Images of FLAG LRRK2
 R1441G and FLAG LRRK2 R1441GK17AK18A expressing HeLa cells. Images from
 three different experiment per condition.
 <strong>-Figure6C</strong>_ Prism files containing
 tabular data of quantification by Mander’s Coefficient colocalization
 analysis. <strong>Figure7 zip
 file:</strong>
 <strong>-Figure7RAWdata</strong>_ 700 and 800
 channels of blots for figure7ABCD.
 <strong>-Figure7EF</strong>_ Prism files containing
 tabular data of quantification of blots of MLi2 washout
 experiments. <strong>Figure8 zip
 file:</strong>
 <strong>-Figure8RAWdata</strong>_ TIRF movies of
 LRRK2 recruitment to Rab10-lipid bilayers.
 <strong>-Figure8A</strong>_ 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. <strong>-Figure8B</strong>_ Prism
 files containing tabular data of quantification of R1441G LRRK2
 recruitment to noRab10 or Rab11-lipid bilayers fitted to a non-linear
 regression curve.
 <strong>-Figure8C</strong>_ 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.
 <strong>Figure8S2</strong>_ 700 and 800 channels of
 blots for figure for figure 8S2. <strong>Figure9
 zip file:</strong>
 <strong>-Figure9RAWdata</strong>_ 700 and 800
 channels of blots for figure9AB.
 <strong>-Figure9CD_</strong> Prism files containing
 tabular data of quantification of blots in figure9AB.