Microscale thermophoresis (MST) is a powerful tool to measure the affinities of interactions between proteins. We present here our method for determining the binding of Rab29 GTPase to the LRRK2 (Leucine rich repeat kinase 2) N-terminal Armadillo domain. Work from several labs has shown that Rab29 can recruit LRRK2 to the Golgi, where it normally resides, or to other compartments, when artificially relocalized to another cellular compartment. MST has enabled us to define the precise binding site for Rab GTPases on the LRRK2 Armadillo domain.

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). 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 domain, 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 demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. 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 membrane activation of LRRK2 kinase activity.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease, and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). 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 domain, 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 demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. 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 membrane activation of LRRK2 kinase activity. Editor's evaluation This article, which is of interest to membrane biologists and colleagues in signal transduction, examines the interesting question of whether LRRK2 recruitment to membranes may regulate its activity. Membrane association involves binding to membrane-tethered Rab GTPases via LRRK2's Armadillo domain, and the authors provide an exciting and elegant feed-forward mechanism to describe how recruitment of phospho-RAB8 can promote phosphorylation of RAB10. https://doi.org/10.7554/eLife.79771.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson’s disease and lead to the phosphorylation of a subset of Rab GTPases (Alessi and Sammler, 2018; Pfeffer, 2022), in particular, Rab8A, Rab10, and Rab29 within a conserved residue of the Switch II effector-binding motif. Rab GTPases are master regulators of membrane trafficking and are thought to serve as identity determinants of membrane-bound compartments of the secretory and endocytic pathways (Pfeffer, 2017). In their GTP-bound forms, Rabs are best known for their roles in linking motor proteins to transport vesicles and facilitating the process of transport vesicle docking. Our previous work showed that Rab phosphorylation blocks the ability of Rab proteins to be activated by their cognate guanine nucleotide exchange factors or to bind to the GDI proteins that recycle GDP-bearing Rabs from target membranes to their membranes of origin (Steger et al., 2016; Steger et al., 2017). Moreover, phosphorylation of Rab8A and Rab10 blocks their ability to bind known effector proteins and enhances binding to a novel set of effectors that includes RILPL1, RILPL2, JIP3, JIP4, and MyoVa proteins (Steger et al., 2017; Waschbüsch et al., 2020; Dhekne et al., 2021). Thus, Rab phosphorylation flips a switch on Rab effector selectivity that can drive dominant physiological changes, including blocking primary cilia formation (Steger et al., 2017; Dhekne et al., 2018; Sobu et al., 2021; Khan et al., 2021) and autophagosome motility in axons (Boecker et al., 2021). Most LRRK2 is found in the cell cytosol where it appears to be inactive (Biskup et al., 2006; Berger et al., 2010; Purlyte et al., 2018). Recent structural analysis of the catalytic, C-terminal half of LRRK2 (Deniston et al., 2020) and full-length human LRRK2 protein yielded structures of both monomeric and dimeric, inactive states (Myasnikov et al., 2021). Several groups have reported that active LRRK2 is a dimer (Greggio et al., 2008; Klein et al., 2009; Sen et al., 2009; Berger et al., 2010; Civiero et al., 2012; Guaitoli et al., 2016), and higher-order forms were detected on membranes upon crosslinking (Berger et al., 2010; Schapansky et al., 2014) and upon Rab29 binding (Zhu et al., 2022). Thus, LRRK2 membrane association is associated with kinase activation; however, the molecular basis for this activation is not yet known. Exogenously expressed, Golgi-localized Rab29 protein can recruit LRRK2 onto membranes and activate it there for both auto- and Rab substrate phosphorylation (Kuwahara et al., 2016; Liu et al., 2018; Purlyte et al., 2018; Madero-Pérez et al., 2018). Indeed, even Rab29 artificially anchored on mitochondria can activate LRRK2 and drive its membrane recruitment (Gomez et al., 2019). McGrath et al., 2021 implicated LRRK2 residues 386–392 as being important for the interaction of Rab29/32/38 family members with the LRRK2 kinase Armadillo domain. However, the LRRK2 Armadillo domain is located at some distance from the kinase domain, at least in the current structure models for LRRK2 protein (Myasnikov et al., 2021). Thus, how Rab29 binding might activate LRRK2 kinase activity is not at all clear. In addition, because Rab29 is not needed for LRRK2 action on Rab8A or Rab10 proteins (Kalogeropulou et al., 2020), other pathways for LRRK2 activation must exist. In this study, we define a specific patch (‘site #1’) of the LRRK2 Armadillo domain that binds to Rab8A, Rab10, and Rab29 protein with affinities similar to those reported previously (McGrath et al., 2021). More importantly, we identify a distinct region of LRRK2 Armadillo domain (‘site #2’) that binds specifically to LRRK2-phosphorylated Rab8A and Rab10 proteins, to establish a feed-forward activation mechanism for membrane-associated LRRK2 kinase. Results Rab29 binds to the C-terminal portion of the LRRK2 Armadillo domain McGrath et al., 2021 showed that the LRRK2 Armadillo domain residues 1–552 contain a binding site that interacts specifically with purified Rab29, 32, and 38 in vitro with affinities of 2.7, 1.2, and 1.2–2.4 µM, respectively. We used microscale thermophoresis to determine the affinity of other Rab GTPase substrates with this portion of LRRK2 kinase. For these experiments, portions of the LRRK2 Armadillo domain were fluorescently labeled and incubated with Rab GTPases in the presence of Mg2+-GTP. Figure 1 shows binding curves for Rab29 with full-length Armadillo domain (residues 1–552, Figure 1A), as well as sub-fragments composed of LRRK2 residues 1–159 (Figure 1C) or 350–550 (Figure 1D). Rab29 showed specific binding to the full-length 1–552 Armadillo fragment with a KD of 1.6 µM (Figure 1A), comparable to that reported previously using other methods (McGrath et al., 2021). Under these conditions, the non-LRRK2 substrate Rab7 protein failed to bind to the Armadillo 1–552 fragment (Figure 1B). No Rab29 binding was detected to a fragment representing the N-terminal 1–159 LRRK2 residues (binding >29 µM; Figure 1C); essentially full binding was observed with a fragment encompassing residues 350–550 (KD = 1.6 µM; Figure 1D). Thus, Rab29 binds to the C-terminal portion of LRRK2’s Armadillo domain at a site that we will refer to as site #1. Figure 1 Download asset Open asset Rab29 binds to the C-terminal portion of the LRRK2 Armadillo domain. Microscale thermophoresis of full-length (residues 1–552), labeled LRRK2 Armadillo domain with His-Rab29 (A) or with His-Rab7 (B). (C, D) Microscale thermophoresis of labeled LRRK2 Armadillo domain residues 1–159 (C) or 350–550 (D) with Rab29. Purified Rab29 was serially diluted and then NHS-RED-labeled-LRRK2 Armadillo (final concentration 100 nM) was added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations. Data are summarized in Table 1. Rab8A and Rab10 bind to the LRRK2 Armadillo domain Similar experiments were carried out with Rab8A and Rab10, the most prominent LRRK2 substrates (Steger et al., 2017). Rab8A-bound full-length Armadillo domain with a KD of 2.9 µM (Figure 2A) showed weaker interaction with the LRRK2 1–159 fragment (KD ~ 6.7 µM; Figure 2B) and good binding to the 350–550 fragment (KD = 2.3 µM; Figure 2C). These data indicate that Rab8A may bind to the same site as Rab29. Like Rab8A, Rab10 bound to full-length Armadillo 1–552 with a KD of 2.4 µM (Figure 2D); weaker binding was detected for 1–159 and 350–550 fragments, yielding KDs of 5.1 µM in both cases (Figure 2E and F). Thus, in addition to Rab32, 38 and 29, Rabs 8A and 10 can bind to LRRK2 residues 350–550. Note that Rab32 and Rab38 are not substrates of LRRK2 kinase as they lack a phosphorylatable Ser/Thr residue in the Switch II motif (Steger et al., 2016; 2107); they show extremely narrow tissue-specific expression but are related to Rab29 protein. Figure 2 Download asset Open asset Rab8A and Rab10 bind to the LRRK2 Armadillo domain. (A–C) Microscale thermophoresis of labeled, LRRK2 Armadillo domain fragments comprised of residues 1–552, 1–159, or 350–550 with Rab8A Q67L as indicated. (C–E) Microscale thermophoresis for Rab10 Q68L (1–181) with indicated LRRK2 Armadillo fragments, as in (A). Purified Rab proteins were serially diluted and then NHS-RED-labeled LRRK2 Armadillo domain (final concentration 100 nM) was added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations. Data are summarized in Table 1. Residues critical for Rab GTPase binding to LRRK2 residues 350–550: Site #1 Previous work implicated LRRK2 residues 386–392 in contributing to a Rab29/32/38 binding interface (McGrath et al., 2021). We used a microscopy-based assay to identify any portions of the first 1000 residues of L. RRK2 that would relocalize to the Golgi upon co-expression with Golgi-localized, HA-Rab29 protein (Figure 3—figure supplement 1). Twenty-two constructs were transfected into cells and their localization scored visually. The smallest fragment of LRRK2 that interacted with HA-tagged Rab29 in HeLa cells, thereby co-localizing at the Golgi complex, encompassed LRRK2 residues 350–550. We next deployed AlphaFold docking (Jumper et al., 2021) using ColabFold (Mirdita et al., 2022) and the AlphaFold2_advanced.ipynb notebook with the default settings to model the interaction of Rab29 with the LRRK2 350–550 fragment (Figure 3A, Figure 3—figure supplement 2). Residues highlighted in red show key contacts between LRRK2 and Rab29 and will be shown below to be essential for detection of this interaction in cells. This modeled structure of site #1 is extremely similar to that of the recently reported experimental cryo-EM structure of Rab29 complexed full-length LRRK2 (Zhu et al., 2022). Figure 3 with 4 supplements see all Download asset Open asset Characterization of critical LRRK2 residues mediating binding to Rab29. (A). Predicted interactions between Rab29 and the LRRK2 Armadillo domain using AlphaFold docking (Jumper et al., 2021), ColabFold (Mirdita et al., 2022), and the AlphaFold2_advanced.ipynb notebook default settings. Residues identified in red show key contacts between LRRK2 and Rab29; orange and yellow coloring indicates the Switch I and Switch II domains of Rab29. (B) The wild-type and indicated mutants of full length of GFP-LRRK2 were co-expressed with HA-Rab29 in HeLa cells. 24 hr post transfection, cells were fixed and localization assessed by confocal microscopy. LRRK2 overlap with Rab29 is presented as a Mander’s coefficient determined using CellProfiler software (McQuin et al., 2018). (C, D) Wild-type and indicated mutants of full length of GFP-LRRK2 (C) or GFP-LRRK2 R1441G (D) were co-expressed with HA-Rab29 in HEK293T cells. 24 hr post transfection, cells were lysed and extracts immunoblotted with the indicated antibodies. Shown are the averages and standard deviations of duplicate determinations; red asterisks indicate preferred mutant. Three metrics were used to evaluate the importance of individual residues to contribute to Rab29 interaction. First, we tested the impact of mutations on the ability of full-length LRRK2 to co-localize with HA-Rab29 at the Golgi in HeLa cells (Figure 3B, Figure 3—figure supplement 3); we also tested the ability of exogenously expressed Rab29 to stimulate activity of the same point mutants in the background of either wild-type LRRK2 (Figure 3C, Figure 3—figure supplement 4A) or pathogenic R1441G LRRK2 (Figure 3D, Figure 3—figure supplement 4B). This work identified four key mutations of highly conserved residues (R361E, R399E, L403A, and K439E) that blocked both the co-localization of LRRK2 and Rab29 in HeLa cells (Figure 3B, red), as well as activation of LRRK2 upon overexpression of Rab29 in HEK293 cells (Figure 3C, red). In experiments undertaken with pathogenic R1441G LRRK2 that is more potently activated by Rab29, the K439E LRRK2 mutation completely blocked LRRK2 kinase activation; R399E showed weak activation (Figure 3D, Figure 3—figure supplement 4B). Some of the other mutants blocked co-localization with Rab29 in HeLa cells without completely suppressing LRRK2 activation following overexpression of Rab29. We therefore recommend using the site#1 K439E LRRK2 mutation to block Rab29 interaction and activation in future work (asterisks in Figure 3B and C) as it shows the lowest amount of Rab29 activation with pathogenic R1441G LRRK2. Altogether, these data highlight the importance of a surface that is comprised of LRRK2 residues Arg361, Arg399, Leu403, Lys439 in binding Rab GTPases (site #1) (Figure 3A, Figure 3—figure supplement 2). Analysis of Rab8A interaction with the LRRK2 350–550 fragment using AlphaFold within ChimeraX 1.4 confirmed the importance of the same LRRK2 residues for Rab8A interaction in silico (Figure 3—figure supplement 2C). PhosphoRab binding to LRRK2: Site #2 To understand the consequences of LRRK2-mediated Rab GTPase phosphorylation, it is important to identify specific binding partners of phosphorylated Rab proteins and study the consequences of such binding events. We recently established a facile method that enables us to monitor phosphoRab binding to proteins of interest in conjunction with microscale thermophoresis binding assays. Briefly, Rab proteins are phosphorylated >90% in vitro by MST3 kinase (Dhekne et al., 2021; Vides and Pfeffer, 2021) that phosphorylates Rab proteins at the same position as LRRK2 kinase (Vieweg et al., 2020). Recombinant MST3 is much easier to purify in large amounts for biochemical experiments than LRRK2. We used this assay to monitor the possible interaction of phosphorylated LRRK2 substrates to the LRRK2 Armadillo domain and were delighted to discover that pRab8A and pRab10 proteins bind with high affinity to a site distinct from that used by non-phosphorylated Rab proteins that we term site #2. As shown in Figure 4, phosphoRab8A and phosphoRab10 bound with KDs of ~900 nM and 1 µM to the full Armadillo domain 1–552 fragment, respectively (Figure 4A and D); this binding reflected interaction with N-terminal LRRK2 residues 1–159 as this fragment was sufficient to yield essentially the same KDs of 1 µM and 700 nM, respectively, for phosphoRab8A and phosphoRab10 proteins (Figure 4B and E). Furthermore, no binding was detected for phosphoRab8A or phosphoRab10 with LRRK2 residues 350–550 (Figure 4C and F). These data demonstrate that Rab8A and Rab10 GTPases, phosphorylated at the same residues modified by LRRK2 kinase, bind very tightly to the LRRK2 N-terminus but no longer interact with the 350–550 region that interacts with dephosphorylated Rab proteins. Figure 4 Download asset Open asset PhosphoRab8A and phosphoRab10 bind with high affinity to the N-terminal portion of the LRRK2 Armadillo domain. (A–F) Microscale thermophoresis of labeled, indicated, LRRK2 Armadillo fragments with His-phosphoRab8A Q67L (A–C) or with His phosphoRab10 Q68L 1–181 (pRab10; D–F). Purified Rab proteins were phosphorylated with Mst3 kinase at 27°C for 2 hr and then serially diluted; NHS-RED-labeled Armadillo (final concentration 100 nM) was then added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations. Note that non-phosphorylated Rab8A and Rab10 also bound to the site #2-containing fragment 1–159 with relatively weak affinities of 5 or 6 µM (Figure 2B and E; Table 1). Interestingly, AlphaFold in ChimeraX (Pettersen et al., 2021) predicts that the 1–159 fragment contains a potential, non-phosphoRab-binding site that is occluded in a longer fragment (1–400), and thus also in full-length LRRK2. Moreover, as discussed below, these KD values may be higher than the concentrations of these Rab GTPases in cells, thus it seems unlikely that non-phosphoRabs interact with site #2 under normal physiological conditions. We conclude that phosphoRab binding is the predominant interaction between LRRK2 1–159 and Rab GTPases. Table 1 Summary of binding affinities. Note that these values are likely underestimates of affinities as typical preparations of the indicated, purified Rab proteins contained ~50% bound GDP and ~50% bound GTP by mass spectrometry. Non-phosphorylated Rab interaction with Armadillo 1–159 is shown in parentheses as it likely reflects binding to an AlphaFold-predicted site near the C-terminus of this fragment that will not be accessible in full-length LRRK2 protein. Armadillo1–159(site #2-containing)Armadillo1–552Armadillo350–550(site #1-containing)Armadillo1–552 K17AArmadillo1–552 K18ARab29>291.6 ± 0.91.6 ± 0.5--Rab10-Q68L(5.1 ±3.1)2.4 ± 0.65.1 ± 2.5--pRab10-Q68L0.71 ± 0.31.0 ± 0.4>29>20>20Rab8A-Q67L(6.7 ± 3.6)2.9 ± 1.22.3 ± 1.0--pRab8A-Q67L1.0 ± 0.60.87 ± 0.4>19.4--Rab7->6.5--- Electrostatic analysis (Jurrus et al., 2018, Pettersen et al., 2004) of a model of the LRRK2 Armadillo domain revealed that the absolute N-terminus of LRRK2 contains a patch of basic amino acids (highlighted in blue) that may comprise a phosphoRab interaction interface (Figure 5A). Such modeling led us to test the role of lysine residues at positions 17 and 18 in mediating LRRK2 interaction. Mutation of either lysine 17 or 18 abolished phosphoRab10 binding to LRRK2 Armadillo domain, with binding decreased to >20 µM upon single mutation at either site (Figure 5C and D). When the conservation score of these residues is analyzed using the Consurf server (Ashkenazy et al., 2016), K17 and K18 have a score or 2 and 8, respectively (9 is the maximum score), indicating that K18 is highly conserved and plays an especially important role. These experiments define a second, Rab binding site #2 that is specific for phosphorylated Rab proteins (Figure 5B). Figure 5 Download asset Open asset Identification of a basic patch at the N-terminus of LRRK2 that is needed for phosphoRab interaction. (A) Electrostatic surface potential of LRRK2 Armadillo domain residues 1–552 modeled using Chimera 2 software (Pettersen et al., 2004); blue indicates a positively charged surface. LRRK2 K17 and K18 are indicated. (B) AlphaFold (Jumper et al., 2021) structure of putative, active LRRK2 with residues that mediate Rab29 binding shown in red (site #1) and the K17/K18 residues that are required for phosphoRab10 binding (site #2) shown in magenta; the kinase domain is shown in blue. (C, D) Microscale thermophoresis of labeled, full-length LRRK2 K17A or K18A Armadillo 1–552 with His phosphoRab10 Q68L 1–181. Purified Rab10 protein was phosphorylated with Mst3 kinase at 27°C for 2 hr and then serially diluted; NHS-RED-labeled Armadillo (final concentration 100 nM) was then added. Graphs show mean and SEM from three independent measurements, each from a different set of protein preparations. To determine the significance of the phosphoRab-binding site in relation to LRRK2 membrane recruitment in cells, we generated full-length FLAG-LRRK2 protein containing point mutations at both lysines 17 and 18 and investigated its cellular localization upon expression in HeLa cells (Figure 6). To improve our ability to detect membrane-associated LRRK2 distribution, cells grown on collagen-coated coverslips were dipped in liquid nitrogen and then thawed in a physiological, glutamate-containing buffer to crack open the plasma membrane and release cytosolic proteins prior to fixation (Seaman, 2004; Purlyte et al., 2018). Under these conditions, LRRK2 co-localizes with phosphorylated Rab proteins (Purlyte et al., 2018; Sobu et al., 2021). Figure 6 Download asset Open asset LRRK2 K17 and K18 are critical for pRab10 interaction in cells. (A) FLAG-LRRK2 R1441G (red) was transfected into HeLa cells plated on collagen-coated coverslips and co-localized with endogenous wild-type pRab10 (green). Cells on coverslips were dipped in liquid nitrogen to deplete cytosol and enhance membrane-bound signal. Insets show enlargements of boxed areas representing peri-centriolar LRRK2 and pRab10. (B) FLAG-LRRK2 R1441G/K17A/K18A (red) was transfected into HeLa cells plated on collagen-coated coverslips and stained and localized with pRab10 (green) as in (A). Scale bars, 10µm. (C) Quantification of pRab10 overlap with LRRK2 by Mander’s coefficient. Error bars represent SEM of means from three different experiments (represented by colored dots), each with >40 cells per condition. Significance was determined by t-test, *p=0.0108. As expected, PhosphoRab10 was detected as a bright spot adjacent to the mother centriole in HeLa cells (green, Figure 6A), and the co-expressed, R1441G pathogenic mutant LRRK2 protein showed good co-localization with phosphoRab10 protein (red, Figure 6A and C), as we have reported previously (Purlyte et al., 2018; Sobu et al., 2021). In contrast, although exogenously expressed, R1441G LRRK2 bearing K17/18/A mutations still led to a perinuclear, phosphoRab10-containing structure (green), LRRK2 (red) displayed much less co-localization with the phosphoRab proteins or with membranes overall (Figure 6B and C). These experiments show that K17 and K18 are important for exogenous LRRK2 membrane association with a pool of highly phosphorylated Rab10 protein. The importance of LRRK2’s N-terminal lysine residues also suggests that caution may be in order when evaluating membrane interactions of LRRK2 tagged N-terminally with larger tags such as GFP that may hinder access to K17/K18. PhosphoRab–LRRK2 interaction increases rates of kinase recovery We next explored the relevance of phosphoRab binding to LRRK2’s N-terminus in relation to the overall kinetics of Rab phosphorylation in cells. LRRK2-mediated Rab GTPase phosphorylation is a highly dynamic process that is counteracted by the action of PPM1H phosphatase (Berndsen et al., 2019); at steady state, only a small fraction of total Rab proteins are LRRK2-phosphorylated (Ito et al., 2016). The initial rate of kinase activity can be determined by monitoring the phosphorylation of Rab10 protein after washout of the LRRK2 inhibitor, MLi-2 (Ito et al., 2016; Kalogeropulou et al., 2020). When HeLa cells were treated with 200 nM MLi-2 for 1 hr and then washed with culture medium, Rab10 was efficiently re-phosphorylated by exogenous, FLAG-tagged, R1441G LRRK2 protein over the 2 hr time course evaluated (Figure 7A and E). In contrast, cells expressing FLAG-R1441G LRRK2 bearing K17/18A mutations showed comparable total phosphoRab10 levels to begin with, but significantly slower re-phosphorylation (Figure 7B and E). Similar results were obtained in experiments comparing the reactivation of FLAG-tagged, wild-type LRRK2 (Figure 7C and F) with that of LRRK2 K17/18A (Figure 7D and F). As reported previously (Ito et al., 2016), wild-type LRRK2 recovery was more efficient than that of R1441G LRRK2. In summary, these experiments demonstrate that K17/K18 residues are important for efficient reactivation of LRRK2 after MLI-2 washout, consistent with their role in anchoring LRRK2 at sites adjacent to phosphorylation substrates. Figure 7 Download asset Open asset LRRK2 K17 and K18 increase endogenous pRab10 recovery after LRRK2 inhibitor washout. (A–D) FLAG-LRRK2 R1441G, FLAG-LRRK2 R1441G/K17A/K18A, LRRK2, or LRRK2 K17A/K18A was transfected into HeLa cells. 48 hr post transfection, cells were treated with 200 nM of MLi-2 for 1 hr. The MLi-2 was then removed by multiple washes and incubated for the indicated times prior to cell lysis. Whole-cell extracts (20 µg) were subjected to quantitative immunoblot analysis using anti-LRRK2, anti-Rab10, and anti-pRab10 antibodies. Numbers at the left of the gels represent the mobilities of molecular weight markers in kilodaltons. (E, F) Quantification of pRab10/total Rab10 fold change and normalized to no MLi2 control. Error bars represent mean ± SD from two different experiments per condition. Figure 7—source data 1 Raw data for gels. https://cdn.elifesciences.org/articles/79771/elife-79771-fig7-data1-v2.zip Download elife-79771-fig7-data1-v2.zip Figure 7—source data 2 Annotated gels. https://cdn.elifesciences.org/articles/79771/elife-79771-fig7-data2-v2.pdf Download elife-79771-fig7-data2-v2.pdf Cooperative LRRK2 membrane recruitment on Rab-decorated planar lipid bilayers Binding of phosphoRabs to site #2 at the N-terminus of LRRK2 (Figure 5B) would set up a feed-forward process whereby the product of an initial phosphorylation reaction would enhance subsequent Rab GTPase phosphorylation by holding the enzyme on the surface of membranes that contain relevant Rab GTPase substrates. To visualize the membrane association process directly, we established a planar lipid bilayer system that would enable us to monitor the interaction of fluorescently labeled, purified, full-length LRRK2 kinase with membrane-anchored Rab10 substrate (Adhikari et al., 2022). For this purpose, bilayers were formed on the surface of glass-bottom chambers comprised of phospholipids of a composition similar to that found in the Golgi (65% DOPC, 29% DOPS, 1% PI(4)P) (Thomas and Fromme, 2016), mixed with 0.01% of the lipophilic tracer DiD dye and 5% DOGS-NTA [Ni2] to enable anchoring of C-terminally His-tagged GFP-Rab10 protein. Binding of fluorescently labeled, hyperactive R1441G LRRK2 was then visualized in real time using total internal reflection (TIRF) light microscopy. Reactions were carried out in the presence of ATP, GTP, and an ATP regenerating system to provide physiological conditions for the full-length LRRK2 enzyme. Note that we routinely utilize R1441G LRRK2 because it is a highly active kinase in cells, although in vitro, R1441G LRRK2 displays the same level of Rab kinase activity as wild-type LRRK2 (cf. Steger et al., 2017). As shown in Figure 8A (red dots), fluorescent R1441G LRRK2 bound efficiently to lipid bilayers only in the presence of pre-anchored Rab10 protein (compare with purple dots in 8B) and not when Rab11 protein was instead employed (Figure 8B, green dots; Videos 1–3). Importantly, almost no binding was observed with kinase inactive D2017A LRRK2 (Figure 8A, yellow dots, Video 4; Steger et al., 2016). This indicates that at least Rab10 GTPase binding to site #1 residues 361–451 results in a low-affinity interaction that is not sufficient to retain this inactive LRRK2 protein on the bilayer under these conditions (7 nM LRRK2, 2.5 µM Rab10). Reactions containing the type I MLi-2 inhibitor showed aggregation of the fluorescent LRRK2 protein, as has been seen in cells. Incubations containing the type 2 inhibitor, GZD-824 (Tasegian et al., 2021), showed weak binding, consistent with a requirement for phosphoRab10 generation to support LRRK2 binding to site #2’s K17 and K18; however, under these conditions, LRRK2 was not monodisperse and could not be analyzed further. Importantly, R1441G LRRK2 mutated at lysines 17 and 18 bound to a lower extent than R1441G LRRK2 (Figure 8A, blue dots; Video 5), confirming their important role in binding to phosphorylated Rab8A and Rab10. It is noteworthy that the K17/K18 mutant protein showed higher binding than the D2017A mutant, suggesting that a non-phosphoRab-binding site may be more accessible for binding in an active versus inactive LRRK2 protein conformation. Figure 8 with 2 supplements see all Download asset Open asset Feed-forward pathway for Rab10 phosphorylation is dependent on LRRK2 kinase activity. (A) Fluorescence intensity traces of individual, single molecules of 7 nM CF633-labeled FLAG-LRRK2 R1441G on a substrate-supported lipid bilayer decorated with lipid-anchored GFP-Rab10 Q68L-His across 600 s of live total internal reflection (TIRF) microscopy. Red, R1441G; blue, K17A/K18A/R1441G; yellow, D2017A. (B) Reactions were carried out as in (A) except Rab10 was omitted (purple) or Rab10 was replaced with Rab11 (green). Dashed lines in (A) and (B) represent time of addition of fluorescently labeled LRRK2 at 60 s; shown are representative experiments carried out at least three times for each condition. Fluorescence intensity was fitted by a nonlinear regression curve for two-phase association. Fold change was calculated by dividing the average fluorescence intensity at steady state and subtracting background fluorescence intensity average determined from 60 s prior to LRRK2 addition. (C) Rate of membrane association of LRRK2 as a function of Rab10 concentration. This curve was fitted by a nonlinear regression fit using PRISM software (MathWorks) to determine a Hill coefficient. Data are from two independent experiments plotted together. Video 1 Download asset Th

Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va's role in ciliogenesis.

Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, "GTP-locked" Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel Rab29 mutants (I64T and V156G) that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation.

Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinson's disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinson's, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here, we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on the assessment of Rab10 and Rab12 phosphorylation, in wild type LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that stimulation of Rab10 and Rab12 phosphorylation induced by agents that stress the endolysosomal system (nigericin, monensin, chloroquine and LLOMe) is suppressed by LRRK2 inhibitors but not blocked in Rab29 deficient cells. From the agents tested, nigericin induced the greatest increase in Rab10 and Rab12 phosphorylation (5 to 9-fold). Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.

Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409, and is degraded during cytokinesis. Mid2 depletion led to astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant astrin in astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis. These results could help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.