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• W2988487066 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. https://doi.org/10.7554/eLife.12813.001 eLife digest Parkinson’s disease is a degenerative disorder of the nervous system that affects approximately 1% of the elderly population. Mutations in the gene that encodes an enzyme known as LRRK2 are the most common causes of the inherited form of the disease. Such mutations generally increase the activity of LRRK2 and so drug companies have developed drugs that inhibit LRRK2 to prevent or delay the progression of Parkinson’s disease. However, it was not known what role LRRK2 plays in cells, and why its over-activation is harmful. Steger et al. used a 'proteomics' approach to find other proteins that are regulated by LRRK2. The experiments tested a set of newly developed LRRK2 inhibitors in cells and brain tissue from mice. The mice had mutations in the gene encoding LRRK2 that are often found in human patients with Parkinson’s disease. The experiments show that LRRK2 targets some proteins belonging to the Rab GTPase family, which are involved in transporting molecules and other 'cargoes' around cells. Several Rab GTPases are less active in the mutant mice, which interferes with the ability of these proteins to correctly direct the movement of cargo around the cell. Steger et al.’s findings will help to advance the development of new therapies for Parkinson’s disease. The next challenges are to identify how altering the activity of Rab GTPases leads to degeneration of the nervous system and how LRRK2 inhibitors may slow down these processes. https://doi.org/10.7554/eLife.12813.002 Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 1–2% of the elderly population (Lees et al., 2009). Environmental and genetic factors contribute to the development of the disease, but its precise etiology still remains elusive (Burbulla and Krüger, 2011). Genome-wide association studies (GWAS) have related 28 genetic risk variants at 24 loci with nonfamilial PD (Nalls et al., 2014). Among those, mutations in LRRK2 (Park8) are also found in hereditary forms, pinpointing a shared molecular pathway driving pathogenesis in both familial and non-familial PD and comprising the most common cause of the disease (Simón-Sánchez et al., 2009; Satake et al., 2009). LRRK2 encodes a large protein composed of central kinase and GTPase (ROC-COR) domains that are surrounded by multiple protein-protein interaction regions. PD pathogenic LRRK2 mutations map predominantly to the kinase (G2019S, I2020T) and the ROC-COR domains (R1441C/G/H, Y1699C), implying that these enzymatic activities are crucial for pathogenesis (Rudenko and Cookson, 2014). Presently, it is unclear how LRRK2 mutations occurring in different functional domains all predispose to PD. The most common PD-associated LRRK2 mutation is the G2019S amino acid substitution, which activates the kinase two- to threefold (West et al., 2005; Khan, 2005; Jaleel et al., 2007). Since protein kinases are attractive pharmacological targets, this finding has raised hopes that selective LRRK2 inhibition can prevent or delay the onset of PD (Yao et al., 2013). Extensive studies of LRRK2 have associated it with diverse cellular processes such as Wnt signaling, mitochondrial disease, cytoskeleton remodeling, vesicular trafficking, autophagy, and protein translation (Taymans et al., 2015; Cookson, 2015; Schapansky et al., 2014; Papkovskaia et al., 2012). Moreover, several LRRK2 substrates have been reported previously; however, evidence that they are phosphorylated by LRRK2 in a physiological context is generally lacking and proofs are confined to in vitro approaches or to cellular systems using overexpressed kinase (Jaleel et al., 2007; Kumar et al., 2010; Ohta et al., 2011; Kawakami et al., 2012; Bailey et al., 2013; Martin et al., 2014; Qing et al., 2009; Chen et al., 2012; Gloeckner et al., 2009; Imai et al., 2008; Gillardon, 2009; Kanao et al., 2010; Matta et al., 2012; Xiong et al., 2012; Yun et al., 2013; Yun et al., 2015; Krumova et al., 2015). Significant off-target effects for LRRK2 compounds that have been used previously further complicate interpretation of the data (Schapansky et al., 2015). Overall, there is little consensus on the cellular roles of LRRK2; thus, identification of definitive and verifiable physiological LRRK2 substrates is considered to be one of the greatest challenges in the field (Schapansky et al., 2015). Besides mutations in LRRK2, other genetic risk variants for PD map to the Park16 locus. Among the five genes within this locus is Rab7L1 (also known as Rab29), which together with LRRK2 increases nonfamilial PD risk. Depletion of Rab7L1 recapitulates the dopaminergic neuron loss observed with LRRK2-G2019S expression and its overexpression rescues mutant LRRK2 phenotypes (MacLeod et al., 2013). Rab GTPases comprise ~70 family members in humans, and they are key players in all forms of intracellular vesicular trafficking events (Stenmark, 2009; Rivero-Ríos et al., 2015). Apart from Rab7L1, several other family members have been associated with PD pathogenesis. For example, mutations in Rab39b (Park21 locus) predispose to PD in humans (Wilson et al., 2014; Mata et al., 2015). Moreover, overexpression of Rab8a, Rab1, and Rab3a attenuate α-synclein-induced cytotoxicity in cellular and animal models of PD, suggesting a functional interplay between Rab GTPases and known PD factors (Cooper, 2006; Gitler et al., 2008). Recently, another PD-connected protein kinase termed PTEN-Induce Kinase-1 (PINK1) has been reported to indirectly control the phosphorylation of a small group of Rabs including Rab8a at Ser111 (Lai et al., 2015). Despite these intriguing links, it is presently unclear whether LRRK2 directly or indirectly modulates Rab GTPases at the molecular level and if so, by which mechanism. High-resolution quantitative mass spectrometry (MS) has become the method of choice for confident identification of in vitro and in vivo phosphorylation events (Roux and Thibault, 2013; Lemeer and Heck, 2009; Olsen et al., 2006). With current MS instrumentation, proteomics can identify tens of thousands of phosphosites (Sharma et al., 2014; Mallick and Kuster, 2010). However, challenges in the phosphoproteomic approaches are to determine functionally relevant residues from these large datasets and to establish direct kinase-substrate relationships. As such, we complement the power of modern phosphoproteomics with parallel genetic, biochemical and pharmacological approaches to establish direct, in vivo LRRK2 substrates. Using fibroblasts derived from two different LRRK2 knock-in mouse lines we identify a subset of Rab GTPases as bona-fide LRRK2 targets. LRRK2 phosphorylates these substrates on an evolutionarily conserved residue situated in their switch II domain both in human and murine cells and in mouse brain. The phosphorylation of Rabs by LRRK2 is direct and strikingly all LRRK2 missense mutations that contribute to PD pathogenesis increase the phosphorylation of at least three Rab GTPases. Further, we establish that different PD pathogenic mutations modulate the interaction with a number of regulatory proteins including guanine dissociation inhibitors (GDI1/2). In this way, LRRK2 regulates the specific insertion of Rab GTPases into target membranes thereby altering their membrane-cytosol equilibrium. Results Identification of LRRK2 substrates in mouse embryonic fibroblasts (MEFs) To search for bona-fide physiological LRRK2 substrates, we performed a dual-phosphoproteomic screening approach using knock-in lines harboring either hyperactive LRRK2 or a LRRK2 variant with wild-type activity but insensitive to a highly selective, newly developed LRRK2 compound. For our first screen (PS1), we generated a mouse model harboring the LRRK2-G2019S substitution that increases kinase activity two- to threefold (Figure 1B). We derived fibroblasts from these animals and treated them with two structurally different LRRK2 inhibitors, GSK2578215A (Reith et al., 2012) or HG-10-102-01 (Choi et al., 2012) (Figure 1A and Figure 1—figure supplement 1A). This screening modality offers three major advantages; first, increased activity of the G2019S-LRRK2 kinase amplifies the chance of finding bona-fide substrates, second, using an isogenic system excludes that measured phosphoproteome changes are due to differences in the genetic background and third, considering only the overlapping population of significantly modulated phosphopeptides of two structurally distinct inhibitors constitutes a very stringent criterion for specifically pinpointing LRRK2 substrates. Figure 1 with 2 supplements see all Download asset Open asset Two unbiased phosphoproteomic screens identify physiological LRRK2 targets. (A) Experimental setup of PS1. LRRK2-G2019SGSK mouse embryonic fibroblasts (MEFs, n=5) were treated with DMSO or each of two structurally distinct LRRK2 inhibitors GSK2578215A or HG-10-102-01 (1 µM for 90 min). (B) LRRK2 immunoprecipitated from either knockout (-/-), wild-type (wt) or LRRK2-G2019SGSK (G2019S) knock-in MEFs was assessed for phosphorylation of Nictide (Nichols et al., 2009) peptide substrate in absence or presence of GSK2578215A (2 μM). Western blot below shows that similar levels of LRRK2 were immunoprecipitated. Error bars are mean ± SD (n=3). (C) Scheme of PS2. The higher affinity of MLI-2 toward wt-LRRK2 allows specific pinpointing of LRRK2 substrates when comparing the phosphoproteomes of wt and A2016T MEFs. (D) Kinase activities of wt (closed circles) and A2016T (open circles) GST-LRRK2 [1326-2527] purified from HEK293 cells were assayed in the presence of the indicated concentration of MLI-2 (n=3). (E) Decreased levels of pS935-LRRK2 in wt MEFs after treatment with 10 nM MLI-2. (F) Heat map cluster of phosphopeptides in PS1 (p<0.005) which are downregulated after treatment with both GSK2578215A and HG-10-102-01. (G) Heat map cluster of downregulated (FDR=0.01, S0=0.2) phosphopeptides in PS2. (H) Venn diagram of overlapping downregulated phosphosites in PS1 and PS2. (Biorep= biological replicate). SD, standard deviation. https://doi.org/10.7554/eLife.12813.003 The second screen (PS2) added another layer of specificity by combining phosphoproteomics with genetics and chemical biology. For this, we used fibroblasts derived from either wt or A2016T-LRRK2 knock-in mice and treated them with the newly developed, highly potent and selective LRRK2 compound MLI-2 ( Fell et al., 2015) The A2016T substitution does not change basal LRRK2 activity but decreases sensitivity to MLI-2 ~10-fold (Figure 1C,D and Figure 1—figure supplement 1B). At a dose of 10 nM, we observed a substantial decrease in phosphorylation of LRRK2-pS935, which is associated with LRRK2 kinase activity (Dzamko et al., 2010), in wt but not in A2016T cells (Figure 1E and Figure 1—figure supplement 1C). Under these conditions, the phosphoproteome of wt MEFs includes both LRRK2-specific and off-target sites, whereas A2016T (which is resistant to MLI-2) only includes off-targets. Therefore, direct quantitative comparison should reveal true LRRK2 substrates (Figure 1C). Using a state-of-the-art workflow for phosphopeptide enrichment, label-free LC-MS/MS and the MaxQuant environment for stringent statistical data evaluation (Humphrey et al., 2015; Cox and Mann, 2008; Cox et al., 2011), we quantified over 9000 high-confidence phosphosites in each replicate in both screens (median R=0.80 and 0.89 in PS1 and PS2, respectively), (Figure 1—figure supplement 1D–G and Supplementary file 1). Independently acquired proteome measurements verified that the detected phosphorylation changes in PS2 were not due to altered protein abundances (changes as determined by label-free quantification in MaxQuant (Cox et al., 2014) were less than twofold [Supplementary file 2]). Next, we determined how many of the identified sites were significantly and robustly modulated. As we were interested in capturing the most strongly regulated sites, we required that the fold change had to be at least as strong as pS935-LRRK2. In PS1, we thus found 234 significantly regulated sites after treatment with each of the two LRRK2 compounds GSK2578215A and HG-10-102-01 (ANOVA, p<0.005), with 78 sites regulated by both (Figure 1—figure supplement 2A). Hierarchical clustering divided them into several subgroups (Figure 1—figure supplement 2B–C and Supplementary file 3A). Besides revealing potential off-target sites of the two LRRK2 inhibitors, this identified a particularly interesting cluster containing 47 sites that were downregulated after treatment with both compounds (Figure 1—figure supplement 2C, cluster 5). In PS2, we identified 204 significantly regulated sites (two sample t-test, FDR=0.01, S0=0.3), when comparing wild-type and inhibitor-resistant LRRK2 fibroblasts, with 128 sites specifically downregulated in the wild type, thus excluding off-target effects (Figure 1G, Figure 1—figure supplement 2D and Supplementary file 3B). Finally, to stringently define LRRK2 substrates, we overlapped the results of the two orthogonal screens. Remarkably, only two phosphosites passed these stringent filtering criteria, our positive control pS935-LRRK2 and the conserved T73 residue of the small GTPase Rab10 (Figure 1H). Direct in vitro phosphorylation of Rab isoforms by LRRK2 Rab10 belongs to the Ras family of small GTPases that regulate intracellular vesicular transport, with ~70 members in human. They function as molecular switches in the tethering, docking, fusion, and motion of intracellular membranes (Stenmark, 2009; Wandinger-Ness and Zerial, 2014). The T73 residue of Rab10 is located in the switch II domain, which is characteristic of Rab GTPases (Figure 2A). This region changes conformation upon nucleotide binding and regulates the interaction with multiple regulatory proteins (Pfeffer, 2005). Sequence alignment revealed that the equivalent site to T73-Rab10 is highly conserved in more than 40 human Rab-family members, indicating strong functional relevance (Figure 2B). Moreover, superposing the crystal structures of multiple Rab GTPases localizes the equivalent residues to T73-Rab10 in nearly the same position (Figure 2—figure supplement 1A). To investigate whether the phosphorylation of Rab10 by LRRK2 is direct, we performed an in vitro kinase assay using recombinant components. Notably, we found that both wt and LRRK2-G2019S, but neither kinase inactive D1994A mutant nor small molecule-inhibited LRRK2, efficiently phosphorylated Rab10, proving a direct kinase-substrate relationship (Figure 2C). Furthermore, incubation of Rab10 with LRRK2 followed by tryptic digestion and MS analysis unambiguously identified T73 as the major phosphorylation site (Figure 2—figure supplement 1B). Given the high conservation of T73-Rab10, we investigated whether other Rab GTPases were also phosphorylated by LRRK2 in vitro. Therefore, we first measured LRRK2-mediated phosphorylation of Rab8a, Rab1a, and Rab1b, all of which contain a Thr at the site equivalent to T73-Rab10, by MS or 32P incorporation followed by Edman sequencing. Remarkably, all proteins were rapidly phosphorylated on the predicted LRRK2 phosphorylation site in the switch II domain (Figure 2D–F and Figure 2—figure supplement 1C–E). Next, we compared Rab family members containing Thr sites in the switch II region with those containing a Ser in the equivalent position. Interestingly, while Rabs with threonines (Rab1b, Rab8a, and Rab10) were efficiently phosphorylated, those with the equivalent serine sites (Rab5b, Rab7a, Rab7L1, Rab12, and Rab39b) were phosphorylated to a drastically lower extent (Figure 2G). This confirms the previously reported in vitro preference for threonines by LRRK2 (Nichols et al., 2009). Finally, we performed a side by side comparison of the phosphorylation efficiencies of recombinant Rab8a and Rab7L1 against two previously reported substrates, moesin (Msn) and Rps15 (Jaleel et al., 2007; Martin et al., 2014). Msn is a cytoskeletal protein and a well-known in vitro LRRK2 substrate, whereas Rps15 is part of the 40S ribosomal subunit and its phosphorylation on Thr136 has been reported to regulate protein translation in D. melanogaster (Martin et al., 2014). In accordance with our previous observations (Figure 2G), phosphorylation levels of RAB7L1 were barely detectable, and even lower than those of Msn and Rps15. Strikingly, levels of pRab8a were about ten times higher as compared to Rps15 and Msn, two of the best in vitro LRRK2 substrates known to date, demonstrating that Rabs with Thr sites in the switch II domain are primary LRRK2 targets (Figure 2H,I). Figure 2 with 1 supplement see all Download asset Open asset Phosphorylation of Rab GTPases by LRRK2 in vitro. (A) Position of threonine 72 in the switch II region of Rab8a (PDB: 4HLY). (B) Sequence alignment of Rab10 and other indicated Rab-family members. (C) Phosphorylation of Rab10 (1 µM) by wt-, G2019S- or kinase inactive LRRK2-D1994A. Inhibition of LRRK2-G2019S by GSK2578215A or HG-10-102-01 prevents phosphorylation. (D) Time course of LRRK2 (wt) mediated Rab8a (4 µM) phosphorylation and (E) quantification of phosphorylation stoichiometry (n=3). (F) Time course of LRRK2-wt-mediated pT75-Rab1a phosphorylation and MS-based label-free quantification (n=3). (G) In vitro phosphorylation of recombinant Rab proteins (4 µM) by LRRK2-wt. (H) Phosphorylation of recombinant Rab7L1, Rab8a, moesin, and Rps15 by LRRK2 and (I) quantification of the signals. For all reactions LRRK2 inhibitors= 2 µM and LRRK2= 100 ng. Error bars indicate mean ± SEM of replicates. MS, mass spectrometry; SEM, standard error of the mean; wt, wild type. https://doi.org/10.7554/eLife.12813.006 A subset of Rabs are physiological LRRK2 substrates Because of the high conservation of T73-Rab10 (Figure 2B) and the ability of LRRK2 to phosphorylate multiple Rabs in vitro, we inspected our quantitative MS data further to determine whether all sequence and structurally equivalent sites are targets of LRRK2. This turned out not to be the case as pS72-Rab7a was not regulated in either of our screens. LRRK2 thus phosphorylates only a subset of Rab GTPases in mouse fibroblasts. Surprisingly, we noticed that pS105-Rab12, which is not phosphorylated by LRRK2 in vitro (Figure 2G), was among the significantly modulated sites in PS1 and also downregulated upon MLI-2 treatment in wt cells as compared to the inhibitor-resistant A2016T mutant in PS2 (Figure 3A,B). However, because of elevated intergroup variability and stringent FDR cut-offs, it was not selected in our first analysis. LRRK2 is found also in lower eukaryotes such as C. elegans and D. melanogaster (Liu et al., 2011) and T73-Rab10 is conserved in these organisms as well. Also, S105-Rab12 is present throughout the vertebrates (Figure 3A,B). We identified both pT73-Rab10 and pS105-Rab12 multiple times with high identification and phosphosite localization scores (Supplementary file 1) and the MS/MS fragmentation spectra of the corresponding synthetic peptides independently validated the MS results (Figure 3—figure supplement 1A,B). Total protein levels of Rab10 and Rab12 did not change appreciably in the A2016T knock-in model as judged by quantitative MS analysis, ruling out that that the observed phospho-level changes are due to differential protein expression (Figure 3—figure supplement 2A). Figure 3 with 3 supplements see all Download asset Open asset A number of Rab GTPases are physiological LRRK2 substrates. (A) MS-quantified pT73-Rab10 peptide intensities in PS1 and PS2. Sequence alignment of the T73-Rab10 region is shown below. (B) Same as (A) with pS106-Rab12. Western blots illustrating phosphorylation of T73-HA-Rab10 (C), S106-HA-Rab12 (D), and T72-Rab8 (E) after induction of LRRK2 expression by doxycycline (1 µg/ml). HG-10-102-01 (1 µM) was added prior to lysis. (F) Western blot of homogenized brain lysates from LRRK2-G2019SLilly mice injected with vehicle (40% HPβCD) or with 3 mg/kg MLI-2 (Biorep= biological replicate) and (G) MS-based quantification of pT72-Rab8 and pS105-Rab12 peptides. (H) Cytoscape network analysis of Rab8a interacting proteins determined by affinity-purification mass spectrometry (AP-MS). LRRK2 is in purple and dashed lines in grey show experimentally determined interactions from string database (http://string-db.org/). https://doi.org/10.7554/eLife.12813.008 To extend the analysis of LRRK2-mediated phosphorylation of Rab10, we used human embryonic kidney cells harboring doxycycline-dependent gene expression of LRRK2-G2019S (HEK293-t-rex-flpIn). Expression of the kinase, treatment with either GSK2578215A or HG-10-102-01 and enrichment of Rab10 by immunoprecipitation followed by quantitative MS analysis confirmed a strong, LRRK2-dependent decrease of pT73-Rab10 peptide levels (Figure 3—figure supplement 2B). Polyclonal antibodies recognizing pT73-Rab10 and pS106-Rab12 (note that the equivalent site is S105 in mouse) independently verified LRRK2-dependent phosphorylation of both Rab isoforms in HEK293 cells (Figure 3C,D). Next, we evaluated whether more Rab isoforms can be phosphorylated in a LRRK2-dependent manner in human cells, focusing on Rab1a, Rab3a, and Rab8a, all of which contain Thr as predicted LRRK2 phosphorylation site (Figure 2B). Therefore, we first ectopically expressed LRRK2 along with either Rab1a or Rab3a, in presence or absence of HG-10-102-01 and quantified pT75-Rab1a and pT86-Rab3a peptide levels by MS. Whereas T86-Rab3a is clearly a LRRK2 target, Rab1a is not, indicating that overexpression of LRRK2 is not sufficient to phosphorylate all Rabs in cells (Figure 3—figure supplement 2C,D). Next, we inhibited LRRK2 in HEK293-t-rex-flpIn cells expressing LRRK2-G2019S and quantified pT72-Rab8. Again, we found a strong decrease of pT72 peptide levels upon LRRK2 inhibition with both GSK2578215A and HG-10-102-01 (Figure 3—figure supplement 2E). An antibody raised for specific detection for pT72-Rab8 confirmed these results further (Figure 2E). To analyze LRRK2-dependent phosphorylation of Rabs in an endogenous context, we quantified pT72-Rab8 and pT73-Rab10 peptide levels in MEFs derived from LRRK2 knockout, wt, or G2019SGSK animals. In the knock-out, the decrease was only about twofold compared to wt, implying a very low intrinsic LRRK2 activity in cells. Consistent with the two- to threefold increased in vitro activity of MEFs-extracted LRRK2-G2019SGSK (Figure 1B), our quantitative MS analysis revealed a threefold increase in both pT72-Rab8 and pT73-Rab10, which was restored to near wt levels by selective LRRK2 kinase inhibition (Figure 3—figure supplement 2F–I). Finally, we globally measured the brain phosphoproteome of LRRK2-G2019SLilly mice injected with vehicle (40% HPβCD) or with MLI-2 (3 mg/kg). Levels of pT72-Rab8 and pS105-Rab12 were decreased more than twofold upon LRRK2 inhibition, validating our findings in the context of a LRRK2 pathogenic mouse model (Figure 3F,G). Having identified Rabs as physiological substrates of LRRK2, we next asked if kinase and substrate also stably interact. Indeed, affinity-purification mass-spectrometry (AP-MS) showed that transiently expressed epitope-tagged LRRK2 and Rab8a efficiently associated with each other, demonstrating that LRRK2 is able to form stable complexes with Rab GTPases in cells (Figure 3H and Figure 3—figure supplement 3A,B). Similarly, Rab10 as well as Rab12 associate with LRRK2 when transiently overexpressed in HEK293 cells (Figure 3—figure supplement 3C–E). Parkinson’s disease-associated pathogenic mutations modulate Rab GTPase phosphorylation levels Pathogenic PD LRRK2 mutations predominantly map to the kinase and the ROC-COR (GTPase) domains and a PD risk factor coding mutation is also found in the WD-40 domain (Martin et al., 2014; Farrer et al., 2007) (Figure 4A). Because it is presently unclear how mutations occurring in distinct LRRK2 functional domains lead to similar disease phenotypes, we decided to investigate if different LRRK2 pathogenic mutations might impact on the phosphorylation status of Rab GTPases. For this, we expressed different disease causing LRRK2 variants along with either Rab8a or Rab10 in HEK293 cells. This revealed that besides PD-associated mutations located in the kinase domain that augment LRRK2 kinase activity, those occurring in the GTPase (ROC-COR) or the WD-40 domains also increased pT72-Rab8a and pT73-Rab10 levels in cells (Figure 4B–E). Figure 4 Download asset Open asset LRRK2 pathogenic variants increase phosphorylation of Rab GTPases. (A) Scheme of LRRK2 and common PD-associated amino acid substitutions (in red). (B) Different LRRK2 versions were co-expressed with Rab8a in HEK293 cells, lysates subjected to immunoblot analysis and (C) indicated signals quantified. (D) and (E) Same as (B) but HA-Rab10 was used. (F) In vitro phosphorylation of recombinant Rab8a (4 µM) by indicated LRRK2 variants (100 ng) and (G) quantification of the signals. HG-10-102-01= 2 µM. Error bars indicate mean ± SEM of replicates (n=3). PD, Parkinson's disease; SEM, standard error of the mean. https://doi.org/10.7554/eLife.12813.012 To determine whether this interplay between different functional domains was direct, we next tested whether pathogenic LRRK2 mutations which lie outside the kinase domain also increase Rab phosphorylation in vitro. As expected, compared to wt, the G2019S mutation resulted in a two- to threefold increase in Rab8a phosphorylation. However, the ROC-COR domain R1441C mutation failed to do so, which is consistent with previous data suggesting that these mutations do not directly enhance LRRK2 kinase activity (Nichols et al., 2010), indicating that its effect on Rab8a phosphorylation levels is mediated by accessory factors in cells (Figure 4F,G). LRRK2 controls the interaction of Rabs with regulatory proteins Rab GTPases consist of a similar core structure comprising highly conserved P-loop, switch I and switch II regions (Pfeffer, 2005). They cycle between the cytosol, in which they are GDP bound and inactive and specific membrane compartments, where they are activated by GDP/GTP exchange (Hutagalung and Novick, 2011). In the crystal structure of Rab8a (Guo et al., 2013), the LRRK2-mediated phosphorylation site is in the switch II region (Figure 2A), which regulates hydrolysis of GTP and coordinates the binding to various regulatory proteins (Pfeffer, 2005). We therefore tested whether the phosphomimetic T72E substitution would modulate GDP/GTP binding or interfere with Rab8a protein interactions. Binding affinities of wt and the TE mutant, determined with fluorescently labeled (N-Methylanthraniloyl, mant) GDP and non-hydrolysable GTP analogue GMPPNP, did not differ (Figure 5—figure supplement 1). In contrast, AP-MS revealed that a number of proteins preferentially bind to non-phosphorylatable T72A-Rab8a compared to the T72E phosphomimetic protein (Figure 5A,B). These were Rab GDP dissociation inhibitors α and β (GDI1 and GDI2), Rab geranyltransferase complex members (CHM, CHML, and RabGGTA/RabGGTB), the Rab8a guanine nucleotide exchange factor (GEF) Rabin8 (Rab3IP), a guanine nucleotide activating protein TBC1D15 and the inositol phosphatase INPP5B (Figure 5C). Figure 5 with 2 supplements see all Download asset Open asset LRRK2 controls the interaction of Rabs with regulatory proteins. (A) Volcano plots showing interactors of GFP-Rab8a (T72A) transiently expressed in HEK293 cells and (B) Proteins differentially binding to T72A as compared to T72E. (C) Fold changes (T72A/T72E, n=4) of regulated proteins shown in (B). (D) Kinetic measurements of the dissociation of mant-GDP from non-phosphorylated and T72 phosphorylated Rab8a by Rabin8. Observed rate constants (kobs) are indicated for each reaction and data points represent mean (n=3). (E) Measurements of mant-GDP dissociation from LRRK2 phosphorylated Rab8a by Rabin8 in absence or presence of λ-phosphatase (λ-PPase) or MLI-2 (1 µM). Error bars are mean ± SD of replicates. https://doi.org/10.7554/eLife.12813.013 Rabin8 interacts with membrane-bound Rab8a and activates it by catalyzing the exchange of GDP to GTP (Westlake et al., 2011). This in turn triggers retention of Rab effector proteins that mediate downstream vesicul" @default.
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• W2988487066 title "Author response: Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases" @default.
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