Activating mutations in the large, multidomain, 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; Vides et al., 2022; Pfeffer, 2023), particularly Rab8A and Rab10 (Steger et al., 2016; Steger et al., 2017). Rab GTPases function in all steps of membrane trafficking by binding to specific effector proteins in their GTP-bound states (Pfeffer, 2017); they are well known for linking motor proteins to transport vesicles and facilitating the transport vesicle docking process.

LRRK2 phosphorylates a single threonine or serine residue in substrate Rab GTPase switch II domains, and this modification blocks the ability of Rabs to be activated by their cognate guanine nucleotide exchange factors, recycled by GDI protein, or bind to their effector proteins (Steger et al., 2016; Steger et al., 2017). Instead, phosphorylated Rabs bind to a new set of phosphoRab effectors that include RILPL1, RILPL2, JIP3, JIP4, and MyoVa proteins (Steger et al., 2017; Waschbüsch et al., 2020; Dhekne et al., 2021). Although only a small percentage of a given Rab protein is LRRK2 phosphorylated at steady state (Ito et al., 2016), binding to phosphoRab effectors has a dominant and powerful effect on cell physiology and can interfere with organelle motility in axons (Boecker et al., 2021), primary ciliogenesis (Dhekne et al., 2018; Sobu et al., 2021; Khan et al., 2021), and centriolar cohesion (Lara Ordóñez et al., 2021).

We have identified a feed-forward pathway that recruits LRRK2 to membranes and can hold it there to enhance subsequent Rab GTPase phosphorylation (Vides et al., 2022). As described in greater detail below, the large multidomain LRRK2 kinase relies on its N-terminal Armadillo domain to associate with membranes. The Armadillo domain contains two substrate Rab binding sites that recruit and anchor LRRK2 on membranes: one for non-phosphorylated Rab proteins and another that can bind LRRK2-phosphorylated Rab8A and Rab10. The presence of two binding sites increases the avidity of LRRK2 for membranes and holds the kinase on membrane surfaces to facilitate subsequent Rab phosphorylation (Vides et al., 2022).

We present here an unbiased, genome-wide CRISPR screen in mouse NIH-3T3 cells undertaken to identify regulators of the LRRK2 pathway. Of the multiple positive and negative hits identified, Rab12 was the most potent regulator of LRRK2 activity when either depleted from cells or overexpressed. We show further our surprising discovery of a third LRRK2 Rab12 binding site in the Armadillo domain that includes residues E240 and S244; site #3 mutations predicted to block Rab12 binding fail to bind Rab12 and show decreased phosphoRab10 levels, consistent with a critical role for Rab12 in LRRK2 activation.

The pooled CRISPR screen to identify modulators of LRRK2 activity utilized mouse NIH-3T3 cells in conjunction with the pooled Brie guide RNA (gRNA) mouse library consisting of 78,637 gRNAs targeting 19,674 genes and an extra 1000 control gRNAs. (A highly detailed protocol can be found on protocols.io; Dhekne et al., 2022a). Briefly, a pooled ‘library’ of Cas9-expressing cells is first generated, each cell harboring a different gene knockout. Genes encoding negative regulators of the LRRK2- phosphoRab10 pathway will increase phosphoRab10 staining when knocked out, and genes encoding positive regulators will decrease phosphoRab10 when knocked out. Fixed cells are stained with an antibody that specifically and sensitively detects phospho-Thr73-Rab10 (hereafter referred to as phosphoRab10) and then sorted by flow cytometry to separate cells based on phosphoRab10 content. Gene knockouts responsible for changes in phosphoRab10 levels are then identified by genomic sequencing of cells with higher or lower than normal phosphoRab10 levels.

Figure 1A shows an example of flow cytometry of anti-phosphoRab10 stained, control mouse NIH-3T3 cells analyzed under baseline conditions (blue) in relation to MLi-2-treated, LRRK2-inhibited cells (green), secondary antibody-only-stained cells (black dashed line), or LRRK2-hyperactivated, nigericin-treated NIH-3T3 cells (pink; Kalogeropulou et al., 2020). The flow cytometry resolution of cells with differing phosphoRab10 levels enabled us to collect the highest 7.5% phosphoRab10 signal and lowest 5% signal and compare these enriched cell populations with unsorted cells. Critical to the success of this method is the ability to obtain non-clumped cells after antibody fixation; otherwise, the average fluorescence of clumps will obscure true hits.

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Statistical analysis of sequencing data from the cells with the lowest phosphoRab10 signal confirmed the success of the screen in that loss of Lrrk2, Rab10, and the Rabif Rab10 chaperone gene (Gulbranson et al., 2017) had the most significant impact on phosphoRab10 expression, as would be expected (Figure 1B and Figure 1—figure supplement 1). Similarly, loss of the Chm gene that is needed for Rab prenylation also led to decreased phosphoRab10. Independent revalidation of the most significant top hits in NIH-3T3 cells (Figure 1C–E and Figure 1—figure supplement 2) by creating individually knocked out cell lines confirmed most of them, and as will be described below, revealed an unexpected role for Rab12 GTPase.

In addition to Rab12, knockout of genes, including Myh9, Cert1, Sptlc2, Ppp2r2a, Ppp1r35, and Nudcd3, also decreased phosphoRab10 intensity by immunofluorescence microscopy, suggesting that the corresponding gene products are also positive regulators of LRRK2 function (Figure 1B–D and Figure 1—figure supplement 1). ER-localized SPTLC2 (serine palmitoyl transferase) is the rate-limiting enzyme in ceramide synthesis, and CERT1 is critical for ceramide transfer from the ER to the Golgi complex. How ceramide synthesis and transport relate to LRRK2 activity will be addressed in future work; chemical inhibition of SPTLC2 with myriocin did not yield a similar phenotype, suggesting that the role of this pathway in phosphoRab10 regulation may be more complex. PPP2R2A was shown previously to similarly influence phosphoRab10 levels in a phosphatome-wide screen to identify phosphoRab10 phosphatases (Berndsen et al., 2019). PPP1R35 was not tested in that screen, but like MYH9, it is involved in primary cilia assembly, and their pericentriolar localizations suggest a connection with phosphoRab10 biology. NUDCD3 stabilizes the dynein intermediate chain and is likely important for concentrating phosphoRab10 at the mother centriole (Zhou et al., 2006; Cai et al., 2009). Finally, 14-3-3 proteins such as YWHAE are known to bind LRRK2 via pSer910 and pSer935 (Nichols et al., 2010) and may stabilize LRRK2 protein.

Knockout of several genes hyperactivated LRRK2 activity and phosphoRab10 levels: these include Atp6v1A, Atp6v0c, Hgs, Phb2, Atp5c, and Csnk2b (Figure 1B, C and E and Figure 1—figure supplement 1). The ATP6 proteins are non-catalytic subunits of the vacuolar ATPase needed for lysosome acidification; their deletion presumably has similar effects as bafilomycin that greatly increases LRRK2 activity (Wang et al., 2021). HGS is also known as HRS and is part of the ESCRT-0 complex; loss of HRS function interferes with autophagic clearance and causes ER stress (Oshima et al., 2016). PHB1/2 are inner mitochondrial membrane mitophagy receptors that are required for Parkin-induced mitophagy in mammalian cells (Wei et al., 2017). Work from Ganley and colleagues has shown an inverse correlation between LRRK2 activity and mitochondrial turnover (Singh et al., 2021). ATP5C1 is part of the mitochondrial ATP synthase complex V; casein kinase 1 alpha has been shown to phosphorylate LRRK2 (Chia et al., 2014) but a role for casein kinase 2B is not yet clear. As reported previously by many other groups, lysosomal and mitochondrial stress increased phosphoRab10 levels.

Loss of Rab12 impacts phosphoRab10 generation

Figure 2A compares the levels of endogenous phosphoRab10 and total Rab10 in parental NIH-3T3 cells, parental cells treated with MLi-2 LRRK2 inhibitor, and a pooled NIH-3T3 cell line in which Rab12 has been knocked out. Quantitation of these data confirmed a roughly fivefold decrease in phosphoRab10 levels under these conditions (Figure 2B). This was entirely unexpected as prior studies on Rab29, a protein that can activate apparent LRRK2 activity under conditions of protein overexpression (Liu et al., 2018; Purlyte et al., 2018); loss of Rab29 did not alter phosphoRab10 levels in a Rab29 mouse knockout model in any tissue analyzed or derived mouse embryonic fibroblasts (MEFs) (Kalogeropulou et al., 2020). We did not analyze Rab8A phosphorylation as the available antibody detects multiple phosphorylated Rab proteins (Steger et al., 2017).

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To confirm these data in an animal model, we analyzed cells and tissues derived from Rab12 knockout mice generated by the Knockout Mouse Phenotyping Program at The Jackson Laboratory using CRISPR technology (Figure 2—figure supplements 1 and 2). Immunoblotting analysis of MEFs confirmed that the heterozygous and homozygous knockouts expressed the expected 50 or 100% loss of Rab12 protein (Figure 2C). MEFs derived from homozygous knockout animals showed as much as 50% decrease in phosphoRab10 levels as detected by immunoblots from multiple clones (Figure 2D); specificity of the detection method was confirmed upon addition of the MLi-2 LRRK2 inhibitor that abolished all phosphoRab10 signals. PhosphoRab7, the product of LRRK1 action (Hanafusa et al., 2019; Malik et al., 2021), appeared to increase moderately as a function of Rab12 loss (Figure 2E). Various tissues were analyzed for phosphoRab10 changes in LRRK2 heterozygous and homozygous knockout animals. As shown in Figure 2F–H, decreases in phosphoRab10 were detected in the homozygous mouse lung with smaller trends in the large intestine and kidney. Together, these data confirm a role for Rab12 in the LRRK2 signaling pathway that is distinct from that of the previously studied Rab29 protein. We were not able to monitor loss of phosphoRab10 in the brain as phosphoRab10 is more difficult to detect in brain tissue that is enriched in the Rab-specific PPM1H phosphatase (Berndsen et al., 2019). Future work will evaluate the consequences of Rab12 knockout in mouse brain and other organs.

Rab12 overexpression enhances LRRK2 activity

Since loss of Rab12 decreased phosphoRab10 levels, we reasoned that increasing Rab12 should increase phosphoRab10 levels. Indeed, overexpression of GFP-Rab12 in A549 cells led to a tenfold increase in phosphoRab10 levels without changing the levels of LRRK2, PPM1H phosphatase (Berndsen et al., 2019) or total Rab10 (Figure 3A and B). The ability of Rab12 to activate LRRK2 was specific for that GTPase in that exogenous expression of GFP-tagged Rab8A, Rab10, or Rab29 failed to show the same high level of phosphoRab10 increase – Rab29 yielded about a fivefold enhancement while Rab12 was almost twice as effective in HEK293T cells (Figure 3C and D).

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The most common, pathogenic, human LRRK2 mutation is LRRK2 G2019S that displays about twofold higher kinase activity than wild type LRRK2; the R1441C mutation activates kinase activity in cells about threefold (Steger et al., 2016). Cells expressing each of these forms showed increased phosphorylation upon Rab12 expression (Figure 3E and F). It is important to note that Rab12 is a more abundant Rab in most tissues than Rab29; for example, A549 cells contain ~134,000 Rab12 molecules and 25,000 Rab29 molecules per cell. This compares with 5000 copies of LRRK2 and 2.5 million copies of Rab10 (https://copica.proteo.info/#/home). Nevertheless, activation was tested at comparable levels of each Rab protein as monitored using anti-GFP antibodies (Figure 3C).

Rab12 activation of LRRK2 did not require Rab12 phosphorylation as the non-phosphorylatable Rab12 S106A was still capable of activation and a phosphomimetic Rab12 S106E failed to increase LRRK2 phospho S1292 (Figure 3G and H). Phosphorylation state Rab mutants must be used with great caution as we have shown previously that Rab8A and Rab10 TA mutants fail to correctly localize and the TE mutants bind phosphoRab effectors with much lower affinity than their correctly phosphorylated counterparts (Dhekne et al., 2018). Nevertheless, the Rab12 S106A mutant was fully capable of LRRK2 activation.

Similar LRRK2 activation results were obtained using immunofluorescence microscopy to assay phosphoRab10 abundance (Figure 4). The phosphoRab10 generated was present on perinuclear membrane compartments (Figure 4A) as seen previously by many groups (Dhekne et al., 2018; Dhekne et al., 2021; Lara Ordónez et al., 2019). PhosphoRab10 staining disappeared in cells expressing PPM1H but not in cells expressing the catalytically inactive H153D PPM1H (Figure 4A and B). These data were confirmed by immunoblot (Figure 4C and D) and suggest that Rab12 is activating LRRK2 along the same pathway of protein phosphorylation studied previously to date.

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Requirements for Rab12 activation of the LRRK2 pathway

It was possible that Rab12 activated a kinase other than LRRK2 to increase Rab10 phosphorylation. This appears not to be the case as GFP-Rab12 expression enhancement of phosphoRab10 levels was not seen in A549 cells lacking LRRK2 expression (Figure 5A and B). It was possible that exogenous GFP-Rab12 inhibited overall Rab phosphatase activity, leading to an apparent increase in phosphoRab10 levels. This was also ruled out as cells lacking PPM1H displayed full Rab12-induced enhancement of phosphoRab10 levels (Figure 5C and D), about fivefold with or without PPM1H.

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Rab12 activation requires a novel Rab binding site in the LRRK2 Armadillo domain

Previous work has identified specific residues within the LRRK2 Armadillo domain that enable LRRK2 to be recruited to the Golgi by exogenously overexpressed Rab29; these residues support direct Rab29 binding (McGrath et al., 2021; Vides et al., 2022; Zhu et al., 2022). In particular, R361, R399, and K439 contribute to a Rab binding ‘Site #1’ that supports binding to purified Rab29 (K_D = 1.6 µM; Vides et al., 2022; Figure 6). Rab8A binds this LRRK2 350–550 region with a similar affinity (2.3 µM) but Rab10 binds less well (5.1 µM; Vides et al., 2022). A second site at LRRK2’s N-terminus (Site #2, K17/K18) mediates interaction with phosphorylated Rab8A and Rab10 proteins. Rab GTPase binding to either or both sites contributes to LRRK2 membrane association as Rabs are themselves membrane anchored by two covalently attached, 20 carbon geranylgeranyl groups.

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AlphaFold (Jumper et al., 2021) in conjunction with Colabfold in ChimeraX (Mirdita et al., 2022; Pettersen et al., 2004) revealed a third Rab binding site (Site #3) when Armadillo domain residues (1–550) were modeled together with Rab12 (Figure 6B and Figure 6—figure supplement 1). (The Armadillo domain is comprised of residues 1–705; we modeled 1–550 as that portion is biochemically stable and well suited for binding experiments.) The predicted local distance difference test (pLDDT) score (0–100) is a per-residue confidence score, with values greater than 90 indicating high confidence; the top 5 structure models (Figure 6—figure supplement 1) yielded pLDDT scores of 87.6, 87.5, 86.8, 87.4, and 86.4, respectively, consistent with high-accuracy modeling.

Mutagenesis across this putative Site #3 binding interface yielded full-length LRRK2 proteins with decreased overall activity as monitored by phosphoRab10 levels in HEK293 cells expressing the mutant proteins (Figure 7A and Figure 7—figure supplement 1). Note that in these experiments the cells rely only on endogenous Rab12 protein. Mutation of E240 and S244 had the greatest impact on LRRK2 activity; remarkably, mutation of F283 to A increased kinase activity twofold. These data demonstrate that Site #3 sequences are important for overall LRRK2 activity.

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Mutation of LRRK2 Site #3 E240R and S244R predicted to be important for Rab12 binding blocked the ability of exogenous Rab12 to enhance phosphoRab10 levels (Figure 7B and Figure 7—figure supplement 1). Moreover, F283A LRRK2 had twofold higher basal activity but was not activated by exogenous Rab12 significantly more than wild type LRRK2 protein. These data strongly suggest that Rab12 activates LRRK2 by binding to Site #3 within the Armadillo domain.

Extensive previous mutagenesis defined Site #1 as being critical for exogenous Rab29-dependent relocalization of LRRK2 to the Golgi complex and apparent activation (Vides et al., 2022). It was therefore important to assess whether Rab29’s ability to increase phosphoRab10 levels upon overexpression relies upon Site #3. As expected, exogenous expression of Rab29 increased phosphoRab10 levels (albeit to a lower extent than exogenous Rab12 expression; Figure 3C and D). However, mutation of Site #3 residues critical for Rab12-mediated LRRK2 activation (E240 and S244) had no effect on the ability of Rab29 to activate LRRK2 kinase (Figure 7C). Similarly, mutation of Site #1 residues preferentially decreased the ability of Rab29 to activate LRRK2 with little if any change in Rab12 activation (Figure 7D). These experiments show that Rab29 interacts preferentially with Site #1 and demonstrate the Rab12 selectivity of Site #3 for LRRK2 activation.

Rab12 binds LRRK2 Site #3 directly

These experiments strongly suggest that Rab29 and Rab12 activate LRRK2 by two different routes: Rab29 via binding to LRRK2 Site #1 and Rab12 via binding to Site #3. We validated Rab12 direct binding to Site #3 using purified Rab12 and Armadillo domain proteins mutated at either Site #1 (K439E) or Site #3 (E240R). As shown in Figure 8, Rab12 bound as well to the wild type Armadillo domain (Figure 8A, 1.4 µM) as to an Armadillo domain construct bearing a Site #1 mutation (Figure 8B, 1.6 µM) as determined by microscale thermophoresis. In contrast, the Site #3 E240R mutation abolished the interaction, yielding a K_D of >40 µM (Figure 8C). Thus, Rab12 binds tightly and directly to Site #3 in vitro and does not appear to interact with Site #1. Interestingly, the LRRK2 Site #3 F283A mutation that increases kinase activity in cells did not influence Rab12 binding significantly, displaying a K_D of 1.2 µM (Figure 8D).

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Binding of Rab12 to LRRK2 Site #3 was also detected in cell extracts in co-immunoprecipitation experiments. As shown in Figure 8E and F, HA-tagged Rab12 and endogenous Rab12 proteins co-precipitated with FLAG-LRRK2 upon transfection in HEK293T cells. In contrast, significantly less co-precipitation was seen with LRRK2 Site #3 mutant E240R and S244R proteins, with or without exogenous HA-Rab12 expression. Rab12 bound F283A LRRK2 as well as wild type LRRK2 protein, consistent with its binding affinity in vitro.

Rab12 drives LRRK2 activation upon lysosomal or ionophore-triggered stress

As mentioned earlier, under conditions of lysosomal damage, LRRK2 is recruited to lysosomes and participates in the repair of damaged endomembranes (Eguchi et al., 2018; Herbst et al., 2020; Bonet-Ponce et al., 2020). Such stress greatly increases LRRK2 kinase activity (Kalogeropulou et al., 2020). Figure 9A–C show that Rab12 is required for the modest increase in LRRK2 activity seen upon lysosomal damage triggered by 1 mM LLOME addition for 2 hr in NIH-3T3 cells. In MEFs (Figure 9D and E), loss of Rab12 dampened but did not abolish the increase in phosphoRab10 levels, especially at later times. Upon treatment of NIH-3T3 cells with nigericin that also causes mitochondrial stress and is a potent activator of the NLRP3 inflammasome (Figure 9F–H), Rab12 knockout diminished Rab10 phosphorylation to control levels. These findings point to the contribution of Rab12 in regulating LRRK2 activity in lysosome repair.

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Using an unbiased, genome-wide screen, we have discovered an important and unanticipated role for the understudied Rab12 GTPase in LRRK2 kinase regulation. Loss of Rab12 from NIH-3T3 and MEF cells (and possibly also mouse lung tissue) significantly decreased phosphoRab10 levels, and Rab12 overexpression increased phosphoRab10 levels. The phosphoRab10 increase was LRRK2-dependent, Rab12-specific, and seen with both wild type and pathogenic mutant LRRK2 proteins. PhosphoRab10 showed the same subcellular localization seen in prior work with cells expressing hyperactive LRRK2 proteins and was sensitive to the Rab-specific, PPM1H phosphatase, consistent with Rab12 activation being part of the normal LRRK2 phosphorylation pathway. Moreover, the increased phosphoRab10 generated as a consequence of Rab12-mediated LRRK2 activation influenced primary cilia formation as expected for typical LRRK2 activation. Site-directed mutagenesis in conjunction with computational modeling revealed a new Rab binding site (Site #3) within the LRRK2 Armadillo domain that is needed for Rab12 binding and activation and is not engaged by Rab29 to trigger apparent kinase activation.

Figure 6 summarizes our current knowledge of Rab GTPase Armadillo domain interactions. Rab29 and its relatives, Rab32 and Rab38, can bind to Site #1 that includes LRRK2 R361, R399, L403, and K439 residues (McGrath et al., 2021; Vides et al., 2022; Zhu et al., 2022); Rab8A is also able to bind at that location (Vides et al., 2022). PhosphoRab8A and phosphoRab10 interact with comparable high affinity with LRRK2 K17/18 at Site #2 (Vides et al., 2022). This study reveals a third interaction interface on the opposite face of the Armadillo domain (relative to Site #1) that engages Rab12 GTPase. The cryoEM structures of full-length LRRK2 (Myasnikov et al., 2021) or LRRK2 in the presence of Rab29 (Zhu et al., 2022) both show an extended and flexible Armadillo domain that extends away from the kinase center and would be available for Rab GTPase engagement.

What are the roles of these multiple Rab binding sites? Site #1 can interact with overexpressed Rab29 protein and bring the mostly cytosolic LRRK2 kinase to the surface of the Golgi complex, which will lead to apparent activation. With regard to membrane anchoring, since loss of Rab29 has no detectable consequence for Rab phosphorylation (Kalogeropulou et al., 2020), it seems likely that Site #1 can also be occupied by the ubiquitous and more abundant Rab8A or possibly Rab10 GTPases. Site #2 that binds to phosphoRabs will also contribute to the membrane anchoring of LRRK2 kinase (Vides et al., 2022); loss of this site decreased overall LRRK2 membrane association at steady state. Site #3 faces the kinase catalytic domain in the AlphaFold model of a putative active LRRK2 protein (Figure 6 and Figure 6—video 1), and we propose that Rab12 binding to Site #3 holds open the kinase to enable substrate access to the active site. Figure 6—video 1 shows a model of Rab12 (pink) bound to the Armadillo domain overlaid onto the AlphaFold model of full-length LRRK2. This model shows that Rab12 occupancy will push against and clash with sequences adjacent to the kinase catalytic domain (shown in blue); presumably Rab12 binding activates the kinase domain through conformational changes. Given that Rab12’s Ser106 phosphorylation site faces the Armadillo domain as part of this Site #3 protein binding interaction, LRRK2 contains at least one additional, yet to be discovered, substrate binding site that positions the Rab phosphorylation site in the correct orientation for LRRK2 kinase phospho-addition.

Rabs 8A, 10, and 12 do not perfectly co-localize in cells yet they can all interact with LRRK2. One possibility is that LRRK2 binds one Rab in each compartment, independently. If Rab8 recruits LRRK2, Rab8 and phosphoRab8 will both cooperate to hold LRRK2 on a Rab8-enriched membrane surface. How would Rab12 come in? It is important to keep in mind the fact that in an A549 cell with 134,000 Rab12 molecules and ~1 million Rab8A proteins, the 5000 LRRK2 molecules may find a subcompartment that contains both Rab8A or 10 and Rab12, despite different primary localizations for the bulk of these Rab proteins. It is also possible that LRRK2 recruited by a Rab to one membrane compartment can phosphorylate a Rab on an adjacent membrane compartment. Future relocalization experiments such as those that anchor LRRK2 on specific subcellular compartments (Gomez et al., 2019) may shed important light on this interesting question.

Beyond activating LRRK2, little else is known about Rab12 GTPase function. GFP-Rab12 co-localizes with transferrin receptors and the PAT4 amino acid transporter and depletion of Rab12 increases the levels of both of these proteins, leading Fukuda and colleagues to conclude that it functions in membrane protein delivery from the endocytic recycling compartment to lysosomes (Matsui and Fukuda, 2011; Matsui and Fukuda, 2013; Matsui et al., 2011, Matsui et al., 2014). These studies showed further that Rab12 regulates the constitutive degradation of PAT4, indirectly influencing mTORC1 activity by modulating cellular amino acid levels. Later work from McPherson showed that under starvation conditions, the Rab12 guanine nucleotide exchange factor DENND3 is phosphorylated by ULK kinase, enhancing its activity and overall levels of Rab12-GTP (Xu et al., 2015). Future work will investigate the consequences of starvation on Rab12 localization and possible roles in autophagy and ciliogenesis regulation. LRRK2 is recruited to damaged lysosomes such as those seen in cells treated with lysosomotropic agents or the LLOME peptide (Eguchi et al., 2018; Herbst et al., 2020; Bonet-Ponce et al., 2020). As we show here, Rab12 also plays a role in activating LRRK2 in that context, but Rab10 phosphorylation was nevertheless seen in Rab12 knockout MEF cells at later times after LLOME addition.

Pathogenic mutations in LRRK2 kinase cause Parkinson’s disease, and LRRK2 kinase inhibitors are currently in clinical trials in the hopes of benefiting patients (Jennings et al., 2022). This work suggests that small molecules that interfere with Rab12 binding to LRRK2 or other means that decrease Rab12 levels may provide additional avenues to target hyperactive LRRK2 kinase.

All primary data associated with each figure has been deposited in a repository and can be found at https://doi.org/10.5281/zenodo.7633917, https://doi.org/10.5281/zenodo.8035447, and https://doi.org/10.5281/zenodo.7659210.

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   (2023) Zenodo

   Genome wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase.

   https://doi.org/10.5281/zenodo.7633917
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   (2023) Zenodo

   Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase.

   https://doi.org/10.5281/zenodo.8035447
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   (2023) Zenodo

   Genome-wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase.

   https://doi.org/10.5281/zenodo.7659210

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Author details

1. Herschel S Dhekne

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Francesca Tonelli, Wondwossen M Yeshaw and Claire Y Chiang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2240-1230

2. Francesca Tonelli

   1. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States
   2. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing – review and editing

   Contributed equally with

   Herschel S Dhekne, Wondwossen M Yeshaw and Claire Y Chiang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4600-6630

3. Wondwossen M Yeshaw

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – review and editing

   Contributed equally with

   Herschel S Dhekne, Francesca Tonelli and Claire Y Chiang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3134-3458

4. Claire Y Chiang

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Writing – review and editing

   Contributed equally with

   Herschel S Dhekne, Francesca Tonelli and Wondwossen M Yeshaw

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0999-9856

5. Charles Limouse

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Data curation, Software, Formal analysis, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2589-4576

6. Ebsy Jaimon

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6845-2095

7. Elena Purlyte

   MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom

   Present address

   University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7291-1549

8. Dario R Alessi

   1. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States
   2. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2140-9185

9. Suzanne R Pfeffer

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration

   For correspondence

   pfeffer@stanford.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

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  citations for umbrella DOI https://doi.org/10.7554/eLife.87098