© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs228809. doi:10.1242/jcs.228809

RESEARCH ARTICLE LRRK1 phosphorylation of Rab7 at S72 links trafficking of EGFR- containing endosomes to its effector RILP Hiroshi Hanafusa1,*, Takuya Yagi1, Haruka Ikeda1, Naoki Hisamoto1, Tomoki Nishioka2, Kozo Kaibuchi2, Kyoko Shirakabe3 and Kunihiro Matsumoto1,*

ABSTRACT active form and a cytosolic, GDP-bound, inactive form. The active, Ligand-induced activation of epidermal growth factor receptor (EGFR) GTP-bound Rab protein binds to various effectors to regulate initiates trafficking events that re-localize the receptor from the cell membrane trafficking (Hutagalung and Novick, 2011; Stenmark, surface to intracellular endocytic compartments. EGFR-containing 2009). Rab7 (herein referring to Rab7a) is a member of the Rab endosomes are transported to lysosomes for degradation by the family that has been demonstrated to play a crucial role in regulating dynein–dynactin motor protein complex. However, this cargo- endo-lysosomal membrane traffic (Guerra and Bucci, 2016). During dependent endosomal trafficking mechanism remains largely the early-to-late endosome transition, Rab7 is recruited to the uncharacterized. Here, we show that GTP-bound Rab7 is subdomains of early endosomes bearing Rab5, followed by Rab5 phosphorylated on S72 by leucine-rich repeat kinase 1 (LRRK1) at the displacement from the same endosome and the acquirement of endosomal membrane. This phosphorylation promotes the interaction of Rab7-mediated transport capacity (Pfeffer, 2013; Poteryaev et al., Rab7 (herein referring to Rab7a) with its effector RILP, resulting in 2010; Rink et al., 2005). Rab7 regulates the movement of recruitment of the dynein–dynactin complex to Rab7-positive vesicles. endosomes along microtubules in a bi-directional manner by This, in turn, facilitates the dynein-driven transport of EGFR-containing interacting with either of two effectors: the effector RILP, which – endosomes toward the perinuclear region. These findings reveal a recruits the dynein dynactin motor complex driving minus-end mechanism regulating the cargo-specific trafficking of endosomes. transport (Johansson et al., 2007; Jordens et al., 2001), or the effector FYCO1, which recruits the kinesin motor driving plus-end KEY WORDS: LRRK1, EGFR trafficking, Rab7 phosphorylation, transport (Pankiv et al., 2010). However, EGFR-containing Rab7 localization endosomes have been shown to be preferentially transported by the Rab7–RILP complex, moving along microtubules toward the INTRODUCTION perinuclear region (Progida et al., 2007; Vanlandingham and EGFR is activated by EGF at the plasma membrane and transduces Ceresa, 2009). It remains largely unknown how Rab7 selectively signals important for cellular responses, such as growth, interacts with RILP to facilitate this endosomal trafficking of EGFR. differentiation, proliferation and motility (Albeck et al., 2013; Recently, we have demonstrated that the endocytic trafficking of Ceresa and Peterson, 2014; Schlessinger, 2000). Activation of EGFR destined for lysosomal degradation is regulated by LRRK1 EGFR also initiates events leading to its own endocytosis. (Hanafusa et al., 2011; Ishikawa et al., 2012; Kedashiro et al., 2015). Internalized receptors are first associated with early endosomes, LRRK1 is related to the familial Parkinsonism gene product which then mature into late endosomes (Goh and Sorkin, 2013; LRRK2 (also known as Park8) and belongs to the ROCO family of Huotari and Helenius, 2011; Scott et al., 2014). During this proteins, which contain a Ras of complex proteins (ROC) GTPase maturation, EGFR is transported to lysosomes for degradation by domain and a MAPKKK-like kinase domain (Bosgraaf and Van the dynein–dynactin motor protein complex (Driskell et al., 2007). Haastert, 2003). LRRK1 forms a complex with activated EGFR and Recent studies have shown that EGFR signaling occurs not only at is involved in the initiation and maintenance of the dynein-mediated the plasma membrane but also in endosomes after internalization transport of early endosomes containing EGFR. This involvement (Irannejad et al., 2015; Sorkin and von Zastrow, 2009). Thus, of LRRK1 is dependent on its intrinsic kinase activity (Hanafusa endosomal trafficking of EGFR determines the spatiotemporal et al., 2011; Ishikawa et al., 2012; Kedashiro et al., 2015). In the regulation of EGFR signaling (Bakker et al., 2017; Miaczynska, initiation step, LRRK1 phosphorylates CLIP-170 (also known as 2013; Tomas et al., 2014). CLIP1), a microtubule plus-end protein, which facilitates its Small GTPases of the Rab family are critical regulators of interaction with a subunit of dynactin p150Glued (also known as membrane trafficking (Hutagalung and Novick, 2011; Stenmark, DCTN1). This, in turn, stimulates the dynein–dynactin complex- 2009). Rabs switch between a membrane-associated, GTP-bound, driven transport of EGFR-containing endosomes (Kedashiro et al., 2015). Furthermore, we have shown that EGFR regulates LRRK1 kinase activity via tyrosine phosphorylation, which is required for 1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. 2Department of Cell Pharmacology, the proper endosomal trafficking of EGFR (Ishikawa et al., 2012). Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya 466-8550, Phosphorylation of LRRK1 at Y944 results in reduced LRRK1 Japan. 3Department of Biomedical Sciences, Graduate School of Life Sciences, kinase activity. Accordingly, mutation of LRRK1 Y944 into a Ritsumeikan University, Noji-higashi, Kusatsu 525-8577, Japan. phenylalanine residue (Y944F) abolishes EGF-stimulated tyrosine *Authors for correspondence ([email protected]; phosphorylation, resulting in the hyper-activation of LRRK1 kinase [email protected]) activity and enhanced motility of EGF-containing endosomes K.M., 0000-0003-4821-0106 towards the perinuclear region. However, the downstream target of LRRK1 that mediates this long-range movement of

Received 10 December 2018; Accepted 2 May 2019 EGFR-containing endosomes had not been identified. Journal of Cell Science

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In this study, we examined the relationship between LRRK1 suggest that within Rab7, S72 is the major phosphorylation site for and Rab7 in mediating long-range EGFR movement. We show LRRK1. We further confirmed that the S72 site in Rab7 can be that LRRK1 interacts with and phosphorylates Rab7 at a conserved phosphorylated by LRRK1 in vivo. For this purpose, we generated S72 residue located in its switch II region. We demonstrate that an antibody specific for Rab7 phosphorylated at S72 (pS72-Rab7). LRRK1-mediated phosphorylation of Rab7 increases its interaction In immunoblots, we detected a single band recognized by the with its effector RILP, but not with FYCO1, and promotes the anti-pS72-Rab7 antibody when Flag–Rab7 was co-expressed long-range minus-end-directed transport of EGFR-containing with GFP-LRRK1(Y944F) but not with GFP–LRRK1(K1243M) endosomes. Thus, our findings reveal a mechanism by which (Fig. S1A). Furthermore, the S72A mutation abolished the LRRK1 determines the selective interaction of Rab7 with its effector pS72-Rab7 signal even in cells co-expressing LRRK1(Y944F) in a cargo-dependent manner. (Fig. S1A). In addition, we found that anti-pS72-Rab7 antibody recognized only the upper band of Rab7 in a Phos-tag PAGE gel RESULTS (Fig. S1B). Taken together, these results indicate that LRRK1 indeed LRRK1 phosphorylates Rab7 on S72 phosphorylates Rab7 S72 in vivo. We have recently reported that LRRK1 regulates the dynein- mediated transport of EGFR-containing endosomes in a manner LRRK1(Y944F) induces Rab7 localization to EGFR- dependent on its kinase activity (Ishikawa et al., 2012; Kedashiro containing early endosomes et al., 2015). Rab7 also plays an important role in the transport of Active Rab7 predominantly localizes to late endosomes and EGFR-containing endosomes toward the nucleus (Ceresa and Bahr, lysosomes (Bucci et al., 2000). We previously reported that a 2006; Vanlandingham and Ceresa, 2009). We therefore examined the hyper-active LRRK1(Y944F) mutant enhances the long-range possible relationship between LRRK1 and Rab7. We first asked movement of EGFR-containing endosomes toward the nucleus, whether LRRK1 and Rab7 physically interact, and found that which results in their perinuclear clustering (Ishikawa et al., 2012). Flag-tagged LRRK1 co-precipitated with GFP-tagged Rab7 We therefore used confocal fluorescence microscopy to investigate (Fig. 1A, lane 2). Since Rab7 cycles between the GDP-bound the effect of LRRK1(Y944F) on the subcellular localization of inactive and GTP-bound active conformations, we next asked Rab7 after EGF stimulation. The movement of EGFR-containing whether LRRK1 preferentially binds to one of these two forms. endosomes was followed in HeLa S3 cells after cells were treated However, we found that LRRK1 interacted similarly with both with fluorescently labeled Alexa Fluor 647-conjugated EGF Rab7(Q67L), a mutant of Rab7 to which GTP is bound, and (A647–EGF). At 10 min after EGF stimulation of control cells, Rab7(T22N), to which GDP is bound (Fig. 1A, lane 3,4 compared to A647–EGF and endogenous Rab7 were distributed in a fine (small lane 2). Thus, LRRK1 interacts with Rab7 independently of its dots) punctate pattern, but did not colocalize (Fig. 2A,D). However, guanine nucleotide-binding state. at 10 min after EGF stimulation of cells expressing GFP– Next, we asked whether LRRK1 phosphorylates Rab7 in an LRRK1(Y944F), a significant fraction of endogenous Rab7 was in vitro kinase assay with purified recombinant GST–Rab7. We colocalized with A647–EGF in the punctate structures (Fig. 2B,D). found that a hyper-active LRRK1 mutant, LRRK1(Y944F) We have previously demonstrated that EGFR and LRRK1 are (Ishikawa et al., 2012), could phosphorylate GST–Rab7, whereas endocytosed together to the endosomes (Hanafusa et al., 2011). the kinase-inactive mutant LRRK1(K1243M) could not (Fig. 1B, Consistent with this, almost all the GFP–LRRK1(Y944F) lanes 2,3). These results show that Rab7 is a kinase substrate of colocalized with A647–EGF (Fig. 2B). Since most of the EGF is LRRK1. To identify the site(s) within Rab7 that are phosphorylated present in early endosomes soon after EGF stimulation, it would be by LRRK1, we incubated GST–Rab7 with LRRK1(Y944F) or expected that LRRK1(Y944F) induces Rab7 localization in early LRRK1(K1243M) under in vitro kinase conditions and subjected endosomes. To test this possibility, we co-stained the cells with the products to analysis by liquid chromatography-coupled tandem markers for different endocytic vesicles. We found that at 10 min mass spectrometry (LC-MS/MS). Four phosphorylation sites, S34, after A647–EGF stimulation of GFP–LRRK1(Y944F)-expressing T40, S72 and T168, were found in Rab7 (Table S1). To determine cells, a fraction of the Rab7 that colocalized with GFP– which Rab7 site(s) are phosphorylated by LRRK1 in vivo,we LRRK1(Y944F) also overlapped with the early endosomal marker analyzed Rab7 phosphorylation by performing phosphate-affinity EEA1, but not with the late endosomal marker CD63 or the (Phos-tag) polyacrylamide gel electrophoresis (PAGE), which lysosomal marker LAMP1 (Fig. S2A–C). Thus, LRRK1(Y944F) detects phosphorylated Rab7 through its slower migration. When induces the localization of Rab7 in early endosomes. In contrast, LRRK1(Y944F) was co-expressed with wild-type Flag–Rab7 in expression of the kinase-inactive GFP–LRRK1(K1243M) failed HEK293 cells, Rab7 proteins appeared in the upper band of the to induce localization of Rab7 to early endosomes (Fig. 2C,D), Phos-tag PAGE gel (Fig. 1C, lane 2). In contrast, co-expression of suggesting that the ability of LRRK1(Y944F) to induce Rab7 LRRK1(K1243M) did not induce a mobility shift (Fig. 1C, lane 1). localization is due to the hyper-activation of its kinase Thus, LRRK1 kinase activity catalyzes the phosphorylation of activity. Furthermore, we found that when LRRK1(Y944F) was Rab7 in vivo. When co-expressed with LRRK1(Y944F), Flag- expressed, phosphorylated Rab7 at S72 was localized in GFP- Rab7(S72A) did not appear in the upper band (Fig. 1C, lane 8). LRRK1(Y944F)-positive EGFR-containing endosomes (Fig. 2E,F). However, other Rab7 mutants, namely, Flag–Rab7(S34A), (T40A) These results suggest that LRRK1(Y944F) phosphorylates Rab7 S72 and (T168A), still gave a band shift in the Phos-tag PAGE analysis in EGFR-containing early endosomes, leading to the stabilization of (Fig. 1C, lanes 4,6 and 10). These results suggest that Rab7 S72 is its endosomal localization. phosphorylated by LRRK1 in vivo. To confirm that Rab7 S72 is the major site of LRRK1 Depletion of PTEN induces Rab7 localization in EGFR- phosphorylation, we produced GST–Rab7(S72A) and performed containing endosomes an in vitro kinase assay. We found that, in contrast to wild-type GST– Recently, Shinde and Maddika have demonstrated that PTEN Rab7, phosphorylation of GST–Rab7(S72A) by LRRK1(Y944F) dephosphorylates Rab7 at S72, a modification that is required for was undetectable (Fig. 1B, lane 3 compared to lane 6). These results the recruitment of Rab7 to late endosomes induced by the GDP Journal of Cell Science

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Fig. 1. LRRK1 interacts with and phosphorylates Rab7 at S72. (A) LRRK1 interacts with Rab7 in a manner independent of its guanine nucleotide-binding state. HEK293 cells were co-transfected with GFP–Rab7 [wild type (WT), Q67L (QL) or T22N (TN) mutants] and empty vector or Flag–LRRK1. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblotting (IB) with anti-Rab7 antibody. Total lysates were immunoblotted with antibodies as indicated. Data are representative of two independent experiments. (B) LRRK1 phosphorylates Rab7 at S72 in vitro. Cos7 cells were transfected with GFP–LRRK1 [K1243M (KM) or Y944F (YF) mutants], and cell lysates were immunoprecipitated with anti-GFP antibody. The immunopurified LRRK1 proteins were incubated with recombinant GST–Rab7 (WT or S72A mutant) in the presence of [γ-32P]ATP for 20 min at 30°C. Autophosphorylated LRRK1 and phosphorylated Rab7 were resolved by SDS-PAGE (upper panels, 32P). Protein input was confirmed by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments. H.C., heavy chain derived from antibodies. (C) LRRK1 induces phosphorylation of Rab7 at S72 in vivo. HEK293 cells were co-transfected with Flag–Rab7 (WT, T40A, S34A, S72A or T168A mutants) and GFP–LRRK1 [K1243M (KM) or Y944F (YF) mutants], and cell lysates were analyzed by Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies as indicated. Filled and open arrowheads indicate unmodified and phosphorylated Rab7, respectively. Data are representative of three independent experiments. dissociation inhibitor (GDI) protein (Shinde and Maddika, 2016). the absence of stimulation. Consistent with their results (Shinde and Indeed, they showed that in PTEN-depleted cells, Rab7 was Maddika, 2016), we confirmed that colocalization of GFP–Rab7 localized in a diffused cytoplasmic pattern, suggesting that with the late endosomal marker CD63 was reduced in PTEN- phosphorylation of Rab7 S72 prevents its membrane localization depleted cells in the absence of EGF stimulation (Fig. S3A,B). by inhibiting the interaction of Rab7 with GDI (Shinde and Therefore, we examined the effect of PTEN knockdown on the Maddika, 2016). This contradicts our results showing that intracellular distribution of Rab7 after EGF stimulation. When phosphorylation of Rab7 S72 stimulates its endosomal control siRNA-treated HeLa S3 cells were stimulated with localization. However, in the Shinde and Maddika study, the Rhodamine-conjugated EGF (Rh–EGF) for 10 min, GFP–Rab7 effect of PTEN depletion on localization of Rab7 was examined in did not localize to Rh–EGF-positive endosomes (Fig. S4A). In Journal of Cell Science

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Fig. 2. LRRK1(Y944F) induces Rab7 localization to EGFR-containing endosomes. (A–C) HeLa S3 cells were transfected with empty vector (A), GFP– LRRK1(Y944F) (YF) (B) or GPF–LRRK1(K1243M) (KM) (C), as indicated. After 16 h serum starvation, cells were stimulated with A647–EGF (40 ng/ml) for 10 min, and then fixed and immunostained with anti-Rab7 antibody. The boxed regions are magnified in insets. Merge indicates pairs of signals consisting of A647–EGF and Rab7 (A) or GFP–LRRK1 (YF or KM) and Rab7 (B,C). Images are representative of three independent experiments. Scale bars: 10 µm. (D) Quantification of colocalization between Rab7 and A647–EGF. Data are plotted as the percentages of Rab7-labeled A647-EGF-containing vesicles out of the total number of A647–EGF-containing vesicles per cell (25 cells and at least 1600 endosomes obtained for each condition from three independent experiments) and the mean±s.e.m. is shown. ***P<0.001; n.s., not significant (Dunnett’s multiple-comparison test). (E,F) Localization of Rab7 phosphorylated at S72 to EGF-positive endosomes. HeLa S3 cells were transfected with empty vector (E) or GFP–LRRK1(Y944F) (YF) (F), as indicated. After 16 h serum starvation, cells were stimulated with A647–EGF (40 ng/ml) for 10 min, and then fixed and immunostained with anti-pS72-Rab7 antibody. The boxed regions are magnified in insets. Merge indicates pairs of signals from A647–EGF and pS72-Rab7 (E), or GFP–LRRK1(YF) and pS72-Rab7 (F). Images are representative of three independent experiments. Scale bars: 10 µm. contrast, we found that, in PTEN-depleted cells, colocalization cells, colocalization of Rab7 with A647–EGF was significantly of GFP–Rab7 with Rh–EGF increased at 10 min after EGF increased (Fig. 3B,D). These results confirm that PTEN negatively stimulation (Fig. S4B). This is similar to the results observed in regulates the localization of Rab7 to EGFR-containing endosomes. cells expressing LRRK1(Y944F) (Fig. 2B,D). These results Importantly, simultaneous depletion of both PTEN and LRRK1 suggest that depletion of PTEN promotes the localization of completely abrogated Rab7 localization to EGFR-containing Rab7 to EGFR-containing endosomes. endosomes (Fig. 3C,D; Fig. S3A). Thus, LRRK1 and PTEN We further examined the subcellular localization of endogenous target S72 in Rab7 as a kinase and a phosphatase, respectively. Rab7 in PTEN-depleted cells. In control cells, the majority of Taken together, these results suggest that LRRK1 promotes the Rab7 was absent from A647–EGF-positive endosomes at 8 min localization of Rab7 to EGFR-containing endosomes, while PTEN after EGF stimulation (Fig. 3A,D). In contrast, in PTEN-depleted prevents its premature localization. Journal of Cell Science

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LRRK1(Y944F) induced phosphorylation of wild-type Rab7 and the GTP-bound form Rab7(Q67L), but not of the GDP-bound form Rab7(T22N) (Fig. 4B, lanes 2–4). These results indicate that LRRK1 also specifically phosphorylates GTP-bound Rab7. The fact that GTP-bound Rab7 localizes to membranes raised the possibility that LRRK1 phosphorylation of Rab7 GTP-bound form also occurs at the membrane. To test this possibility, we constructed Rab7(CS), where two C-terminal cysteine residues (C205 and C207), prenylation of which is known to be required for Rab7 membrane localization (Seabra, 1998; Wu et al., 2009), are replaced by serine residues. Since Rab7(CS) is not prenylated, it not expected to localize to membranes (Seabra, 1998; Wu et al., 2009). Consistent with this expectation, Flag–Rab7(CS) and Flag–Rab7(Q67L/CS) exhibited a diffuse distribution and did not localize to CD63-positive late endosomes (Fig. S5). We found that expression of LRRK1(Y944F) was unable to induce phosphorylation of Rab7(CS) or Rab7(Q67L/ CS) in Phos-tag analysis (Fig. 4B, lanes 5,6). Thus, membrane localization of Rab7 is required for its phosphorylation by LRRK1. These results suggest that LRRK1 selectively phosphorylates GTP-bound membrane-localized Rab7. Consistent with this, a significant but relatively low proportion of wild-type and GTP- bound Rab7 was phosphorylated by LRRK1(Y944F) (Fig. 4C).

LRRK1 phosphorylation of Rab7 promotes its interaction with RILP Rab GTPases cycle between the cytosol, where they are GDP-bound and inactive, and specific membrane compartments, where they are GTP-bound and active (Hutagalung and Novick, 2011; Stenmark, 2009). Rab7 crystal structure studies have revealed that the switch II region regulates hydrolysis of GTP and coordinates the binding to effector proteins (Wu et al., 2005). We therefore tested whether Rab7 protein interactions are modulated by LRRK1 phosphorylation. RILP is one of the Rab7 effector proteins that mediate downstream vesicular trafficking events (Cantalupo et al., 2001; Jordens et al., 2001). To Fig. 3. Effects of PTEN and LRRK1 depletion on Rab7 localization to EGFR- – investigate the effect of LRRK1 phosphorylation of Rab7 S72 on its containing endosomes. (A C) Localization of endogenous Rab7 to EGF- interaction with RILP, we performed pulldown experiments using positive endosomes. HeLa S3 cells were treated with control siRNA (siControl; A), PTEN siRNA (siPTEN; B) or PTEN siRNA plus LRRK1 siRNA Halo-tagged RILP. Lysates prepared from Cos7 cells expressing (siPTEN+siLRRK1) (C). After 16 h serum starvation, cells were stimulated Halo–RILP were first bound to Magne-HaloTag beads. Then, lysates with A647–EGF (40 ng/ml) for 8 min, and then fixed and immunostained of HEK293 cells expressing GFP–Rab7 with or without GFP- with anti-Rab7 antibody. Examples of colocalization of Rab7 with A647–EGF LRRK1(Y944F) were mixed with the beads. These pulldown are indicated with arrows. Images are representative of three independent experiments revealed that the Rab7–RILP interaction was enhanced experiments. Scale bars: 10 µm. (D) Quantification of colocalization between by LRRK1(Y944F) (Fig. 5A). This effect was ablated in the Rab7 and A647–EGF. Data are plotted as the percentages of Rab7-labeled A647–EGF-containing vesicles out of the total number of A647–EGF-containing phosphorylation-incompetent GFP-Rab7(S72A) mutant (Fig. 5A). vesicles per cell (20 cells and at least 1400 endosomes obtained for each We next examined whether the effect of LRRK1 phosphorylation on condition from three independent experiments) and the mean±s.e.m. is shown. Rab7 interaction was effector specific. FYCO1 is another Rab7 ***P<0.001; n.s., not significant (Dunnett’s multiple-comparison test). effector that binds to the switch II domain of Rab7 (Pankiv et al., 2010). However, we found that in contrast to RILP, LRRK1(Y944F) LRRK1 specifically phosphorylates the GTP-bound form had no effect on Rab7–FYCO1 interaction (Fig. 5B). These results of Rab7 in endosomes suggest that LRRK1(Y944F)-induced phosphorylation of Rab7 The LRRK1 phosphorylation site in Rab7, S72, is located in the specifically increases its interaction with RILP. conserved switch II region (Fig. 4A). It has been recently reported Next, we examined the effect of LRRK1(Y944F) on the that another member of the ROCO family, LRRK2, phosphorylates intracellular distribution of RILP after EGF stimulation. RILP a subset of Rab proteins, including Rab3, Rab8, Rab10, Rab12, functions to maintain Rab7 in the active and membrane-bound state, Rab35 and Rab43, at conserved serine and/or threonine residues in and RILP expression has been reported to cause an accumulation the switch II region (Fig. 4A) (Ito et al., 2016; Steger et al., 2016, of late endosomes around the nucleus (Cantalupo et al., 2001; 2017). The switch II region is known to be relatively disordered in Jordens et al., 2001). We expressed Halo–RILP in HeLa S3 cells the inactive GDP-bound state compared with that in the active GTP- and stimulated them with A647–EGF. At 10 min after EGF bound state (Lee et al., 2009; Pfeffer, 2005; Pylypenko et al., 2018). stimulation, Halo–RILP accumulated near the nucleus and failed Indeed, LRRK2 preferentially phosphorylates GTP-bound Rab to localize to A647–EGF-positive endosomes (Fig. 5C,E). proteins (Liu et al., 2018). Therefore, we examined whether LRRK1 Interestingly, when GFP–LRRK1(Y944F) and Halo–RILP were phosphorylation of Rab7 is also specific for the GTP-bound form. co-expressed, colocalization of A637–EGF with Halo–RILP

Phos-tag analysis revealed that when expressed in HEK293 cells, increased (Fig. 5D,E). These results suggest that LRRK1 Journal of Cell Science

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Fig. 4. LRRK1 phosphorylates GTP-bound Rab7 in endosomes. (A) Sequence alignment in the switch II region of Rab7a and other indicated Rab family members. Identical residues are highlighted with black shading. Red characters indicate phosphorylation sites. (B) LRRK1 phosphorylates GTP-bound Rab7 at membranes. HEK293 cells were co-transfected with Flag–Rab7 [WT, Q67L (QL), T22N (TN), CS or QL/CS mutants] and empty vector or GFP–LRRK1(Y944F) (YF), and cell lysates were analyzed by Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies as indicated. Filled and open arrowheads indicate unmodified and phosphorylated Rab7, respectively. Data are representative of three independent experiments. (C) Quantification of Rab7 phosphorylation. Data represent the percentages of phosphorylated Rab7 relative to the total amount of Rab7 (i.e. the upper band+unmodified band=100%). The intensities of the band in the immunoblots in B were measured using FUSION system. Data are combined from three independent experiments. Error bars represent s.e.m. ***P<0.001; n.s., not significant (Dunnett’s multiple-comparison test).

promotes the recruitment of RILP to EGFR-containing endosomes together, these results suggest that the Rab7–RILP–dynein by enhancing the interaction between Rab7 and RILP. complex is necessary for LRRK1(Y944F)-mediated long-range transport of EGFR-containing endosomes toward the nucleus. LRRK1(Y944F)-induced perinuclear clustering of EGFR-containing endosomes is dependent on dynein, DISCUSSION Rab7 and RILP In this study, we show that LRRK1 phosphorylates the conserved S72 Expression of the hyperactive LRRK1(Y944F) mutant enhances the residue within the switch II region of Rab7. This in turn increases the long-range movement of EGFR-containing endosomes, resulting in association of Rab7 with its effector RILP, resulting in enhanced the accumulation of clustered and enlarged endosomes (Ishikawa minus-end transport of EGFR-containing endosomes through a et al., 2012). To examine this phenomenon further, we asked dynein-dependent mechanism. Recent studies have shown that the whether the effect of LRRK1(Y944F) on the localization of endocytic trafficking of EGFR depends on the concentration of EGF EGFR-containing endosomes is dependent on the dynein motor. to which cells are exposed (Sigismund et al., 2005). When cells are Stimulation of LRRK1(Y944F)-expressing cells with Rh–EGF for stimulated with low EGF concentrations, EGFR is typically subjected 30 min induced the perinuclear clustering of Rh–EGF-positive to clathrin-mediated endocytosis and a large fraction of the receptor is endosomes (Fig. 6A,E). In contrast, pre-treatment of these cells with recycled to the cell surface, allowing for prolonged EGFR signaling ciliobrevin, a specific inhibitor of dynein (Firestone et al., 2012), (Sigismund et al., 2008). In contrast, at higher concentrations of EGF, resulted in the scattered distribution of GFP–LRRK1(Y944F) and the receptor is preferentially internalized by a clathrin-independent Rh–EGF-positive endosomes in the cytosol (Fig. 6B,E). This pathway that involves its ubiquitylation, sorting into the intraluminal result confirmed that LRRK1(Y944F)-mediated clustering of vesicles (ILVs) of the multivesicular body and transfer to the EGFR-containing endosomes is dependent on dynein. lysosomes for degradation (Futter, 1996; Sigismund et al., 2005, We next examined whether Rab7 or RILP is required for 2013). ILV sorting of the receptor also involves the physical removal this process. We found that depletion of Rab7 by means of siRNA of the signaling tail of EGFR from the cytosol, which effectively completely eliminated the LRRK1(Y944F)-induced clustering terminates downstream signaling prior to lysosomal degradation of Rh–EGF-positive endosomes (Fig. 6C,E; Fig. S6A,B). (Eden et al., 2009; Katzmann et al., 2002). These systems are Furthermore, depletion of RILP also significantly inhibited this thought to allow cells to both amplify weak physiological inputs clustering (Fig. 6D,E; Fig. S6A). These results were also observed and to effectively cope with overstimulation. We have previously using different Rab7 and RILP siRNAs (Fig. S6C–E). Thus, both demonstrated that LRRK1 is required for the efficient sorting Rab7 and RILP are required for LRRK1(Y944F)-induced of ubiquitylated EGFR into the ILVs by interacting with STAM1 perinuclear clustering of EGFR-containing endosomes. Taken and Hrs, subunits of the endosomal sorting complexes required for Journal of Cell Science

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Fig. 5. LRRK1 phosphorylation of Rab7 promotes its association with RILP. (A,B) LRRK1 promotes the interaction of Rab7 with RILP but not with FYCO1. Cos7 cells were transfected with Halo–RILP (A) or Halo–FYCO1 (B), and cell lysates were bound to Magne-HaloTag beads. HEK293 cells were co-transfected with GFP–Rab7 (WT or S72A) and empty vector or GFP–LRRK1(Y944F) (YF), as indicated. Lysates from HEK293 cells were mixed with Halo-RILP (A)- or Halo-FYCO1 (B)-bound beads. Complex formation was detected by immunoblotting (IB) with anti-GFP antibody. Total lysates were immunoblotted with antibodies as indicated. Data are representative of three independent experiments. (C,D) LRRK1 recruits RILP to EGFR-containing endosomes. HeLa S3 cells were co-transfected with Halo–RILP and empty vector (C) or GFP–LRRK1(Y944F) (YF) (D). After 16 h serum starvation, cells were stimulated with A647–EGF (40 ng/ml) for 10 min, and then fixed and immunostained with anti-Halo antibody. Merge indicates pairs of signals from Halo–RILP and A647–EGF. Images are representative of three independent experiments. Scale bars: 10 µm. (E) Quantification of colocalization between Halo–RILP and A647–EGF. Data are plotted as the percentages of Halo–RILP-labeled A647–EGF-containing vesicles out of the total number of A647–EGF-containing vesicles per cell (20 cells and at least 1100 endosomes obtained for each condition from three independent experiments). Error bars represent s.e.m. ***P<0.001 (Welch’s t-test). transport (ESCRT)-0 complex, and facilitates the dynein-driven localization of Rab7 into late endosomes in unstimulated cells, transport of EGFR-containing endosomes for lysosomal degradation PTEN depletion actually enhances Rab7 localization to EGF- (Hanafusa et al., 2011; Ishikawa et al., 2012; Kedashiro et al., 2015). positive endosomes in EGF-stimulated cells. Furthermore, it is Therefore, LRRK1 might function in the latter system to difficult to elucidate the role of Rab7 S72 phosphorylation by using efficiently terminate EGFR signaling, thus protecting the cell Rab7(S72E). It is known that newly synthesized GDP-bound Rab from overstimulation. proteins interact with Rab escort proteins (REP1 and REP2), which A previous study has shown that PTEN dephosphorylation of interacts with geranylgeranyl transferase type II (GGTII) and Rab7 S72 is necessary for the GDI-dependent recruitment of Rab7 facilitates Rab protein prenylation and membrane anchoring into late endosomes; this study also showed that a constitutive (Hutagalung and Novick, 2011; Pylypenko et al., 2018; Seabra phosphomimetic mutant Rab7(S72E) fails to localize to the and Wasmeier, 2004). In the case of Rab8, phosphomimetic endosomal membrane and does not interact with RILP (Shinde substitution of the residue corresponding to Rab7 S72 interferes and Maddika, 2016). These results are apparently inconsistent with with its interaction with REP1, REP2 and GGTII, resulting in our present findings. However, while PTEN depletion reduces the defective prenylation and failure to localize to the membrane (Steger Journal of Cell Science

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Fig. 6. Dynein, Rab7 and RILP are required for LRRK1(Y944F)- induced perinuclear clustering of EGFR-containing endosomes. (A–D) Effects of a dynein inhibitor, Rab7 siRNA and RILP siRNA on the LRRK1(Y944F)-induced clustering of EGF- positive endosomes. HeLa S3 cells treated with control siRNA (siCtrl; A,B), Rab7 #1 siRNA (C) or RILP #1 siRNA (D) were transfected with GFP–LRRK1(Y944F) (YF). After 16 h serum starvation, cells were stimulated with Rh–EGF (40 ng/ml) for 30 min, and then fixed. The cells shown in B were pre-incubated with ciliobrevin (50 µM) for 30 min before Rh–EGF stimulation. The position of the nuclei of cell in the images is shown with a yellow dotted line. Images are representative of three independent experiments. Scale bars: 10 µm. (E) Quantification of EGF accumulation in the perinuclear region. Histogram indicates the percentage of cells that have endosomes (>2.0 µm diameter) containing Rh–EGF in the perinuclear region. Data are representative of over 100 cells imaged for each condition from three independent experiments. Error bars represent s.e.m. ***P<0.001 (Dunnett’s multiple-comparison test).

et al., 2016). Similarly, the nascent Rab7(S72E) protein may be indicate that LRRK1 phosphorylates the active GTP-bound form of defective in its interaction with REP1, REP2 or GGTII, resulting in Rab7 at the membrane subsequent to its endosomal delivery by the accumulation of the hypoprenylated protein in the cytosol. GDI. A recent study by Steger et al. has shown that LRRK2 Indeed, Rab7(S72E) exhibits a diffused cytoplasmic distribution phosphorylates a subset of Rab proteins on an evolutionarily (Shinde and Maddika, 2016). In addition, because Rab7(S72E) fails conserved residue in the switch II domain, which, in the case of to interact with its GEF CCZ1, it exists predominantly in the GDP- Rab7, corresponds to S72 (Steger et al., 2016, 2017). This region bound inactive form (Shinde and Maddika, 2016). This shift to the undergoes a conformational change upon GTP binding, and this inactive form would explain why the S72 phosphomimetic conformational change is required for Rab interaction with its substitution interferes with its interaction with RILP. The cycling binding partners (Pfeffer, 2005). Indeed, phosphorylation of Rabs of Rab7 between the cytosolic GDP-bound inactive and membrane- on this residue inhibits their association with a number of regulatory associated GTP-bound active conformations is mediated by proteins, including REP1, REP2, GGTII, GDI1, GDI2 and GEFs interactions with a number of regulatory proteins. Our results (Steger et al., 2016, 2017). In contrast, the two effector proteins Journal of Cell Science

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RILP-L1 and RILP-L2 have been shown to bind preferentially to the Plasmids, mutations and RNA interference LRRK2-phosphorylated Rab8, Rab10 and Rab12 and function in Human LRRK1 was cloned from a cDNA library by RT-PCR (our clone the formation of primary cilia (Dhekne et al., 2018; Steger et al., lacks 27 amino acids at the N-terminus compared with NM_024652), and – – – 2017). These interactions are mediated by highly conserved basic GFP LRRK1, GFP LRRK1(K1243M) and GFP LRRK1(Y944F) were residues in the RILP homology (RH) domain of RILP-L1 and generated as described previously (Ishikawa et al., 2012). Rab7(Q67L), Rab7(T22N), Rab7(S34A), Rab7(T40A), Rab7(S72A), Rab7(T168A), RILP-L2, which are also conserved in RILP. Interestingly, we Rab7(CS) and Rab7(Q67L/CS) were generated by using the QuikChange found that LRRK1 phosphorylation on S72 enhanced the binding of site-directed mutagenesis kit according to the manufacturer’s protocol Rab7 to RILP. In contrast, LRRK1 phosphorylation had no effect (Stratagene, La Jolla, CA) and subcloned into the pEGFP-C1 (Clontech) or on the Rab7–FYCO1 interaction. Thus, the effect of Rab7 S72 pCMV-Flag vectors. Halo–RILP and Halo–FYCO1 were obtained from phosphorylation on the effector interaction is specific for Kazusa DNA Res. Inst. (Promega). siRNA targeting human LRRK1 and RILP. These results raise the possibility that LRRK1-mediated negative control siRNAs were obtained as previously described (Hanafusa phosphorylation of Rab7 recruits the RILP–dynein–dynactin et al., 2011). Pre-validated PTEN siRNA was purchased from Qiagen complex to EGFR-positive endosomes, and this would explain (catalog no. SI00301504) and siRNA for human Rab7 #1 [target sequence: why EGFR-containing endosomes are largely restricted to migrate CGGTTCCAGTCTCTCGGTd(TT)], human Rab7 #2 [target sequence, toward the nucleus. Our findings thus provide the first evidence for GGATGACCTCTAGGAAGAAd(TT)], human RILP #1 [target sequence, GATCAAGGCCAAGATGTTAd(TT)] and human RILP #2 [target cargo-dependent regulation of endosomal trafficking. sequence, GCAGCGGAAGAAGATCAAGd(TT)] were purchased from We have previously reported that LRRK1 facilitates the initiation JBioS. Annealed siRNAs were transfected using RNAiMAX (Invitrogen). of the dynein-driven transport of EGFR-containing endosomes The transfected cells were analyzed 72 h after transfection. It was confirmed Glued through the recruitment of p150 at microtubule plus-ends by using FITC-labeled oligonucleotides that almost all HeLa S3 cells had been phosphorylating CLIP-170 (Kedashiro et al., 2015). Thus, the transfected with the siRNA oligonucleotides. hyperactivation of LRRK1 kinase enhances the motility of EGFR-containing endosomes toward the nucleus and causes the Preparation of GST fusion proteins and kinase assays immature perinuclear clustering of EGFR-containing endosomes, The recombinant proteins GST–Rab7 and GST–Rab7(S72A) were E. coli which cannot fuse with lysosomes (Ishikawa et al., 2012). However, each expressed in the strain BL21-CodonPlus (DE3)-RIPL and – the downstream target of LRRK1 in this process has not been purified using glutathione Sepharose 4B (GE Healthcare) following the manufacturer’s guidelines. All GFP–LRRK1 proteins were expressed in Cos7 identified. Here, we identified Rab7 as a substrate for LRRK1 that cells and immunopurified with anti-GFP antibody (4 μl/sample; 598, MBL) maintains the dynein-mediated transport of EGFR-containing (Kedashiro et al., 2015). Kinase reactions were performed in a final volume of endosomes along microtubules. These results suggest that LRRK1 20 µl buffer consisting of 50 mM HEPES pH 7.4, 5 mM MgCl2,5mM 32 plays an important role in the initiation and maintenance of the MnCl2, 0.5 mM DTT, 5 µCi of [γ- P]ATP and 100 µM ATP. Samples were dynein-mediated transport of EGFR-containing endosomes by incubated for 20 min at 30°C and the reactions terminated by addition of phosphorylating CLIP-170 and Rab7, respectively. Laemmli sample buffer and boiling. Samples were resolved by SDS-PAGE LRRK2 has been reported to be present at multiple distinct and analyzed by autoradiography. membrane compartments and to function in vesicle trafficking through the endolysosomal and autophagic pathways (Alegre- Determination of LRRK1 phosphorylation sites of Rab7 Abarrategui et al., 2009; Biskup et al., 2006). Interestingly, by LC-MS/MS LRRK2 also phosphorylates the GTP-bound membrane-associated For LRRK1 phosphorylation site analysis, immunoprecipitated Rab7 proteins were subjected to a non-radioactive in vitro kinase assay and then eluted with form of Rabs (Liu et al., 2018). LRRK1 and LRRK2 might regulate guanidine solution (50 mM NH4HCO3, 7 M guanidine-HCl), followed by different aspects of membrane trafficking by phosphorylating target reduction, alkylation, demineralization and concentration as described Rabs in different contexts. Since active GTP-bound Rabs function by previously (Kedashiro et al., 2015). Rab7 proteins were digested with recruiting distinct sets of effector proteins, it is critical to understand trypsin for 16 h at 37°C. From these peptide samples, phosphopeptides were how they recognize their effectors specifically. Thus, the binding of enriched and captured using the Titansphere Phos-TiO Kit according to the effectors to the Rabs is regulated not only by guanine nucleotide manufacturer’s instructions. Nano-electrospray tandem mass analysis was cycling, but also by LRRK1- and LRRK2-mediated phosphorylation. performed using a Q Exactive mass spectrometer (ThermoFisher Scientific Inc.) system combined with a Paradigm MS4 HPLC system (Michrom BioResources Inc.). Samples were injected into the Advance nanoflow MATERIALS AND METHODS UHPLC/HTS-PAL system equipped with a MonoCap C18 Nano-flow Cell cultures, antibodies and reagents column 0.1 mm×150 mm (GL Sciences). Reversed-phase chromatography HeLa S3, Cos7 and HEK293 cells were cultured in Dulbecco’smodified was performed with a linear gradient (0 min, 5% B; 70 min, 40% B) of Eagle’s medium (DMEM) containing 10% fetal bovine serum. These cell solvent A (H2O with 0.1% formic acid) and solvent B (acetonitrile) at an lines were obtained from the Japanese Collection of Research Bioresources estimated flow rate of 400 nl/min. Ionization was performed with an (JCRB) or the American Type Culture Collection (ATCC) and were regularly ADVANCE CaptiveSpray Source (Michrom BioResources Inc.). A precursor tested for mycoplasma contamination. Antibody against pS72-Rab7 was ion scan was carried out using a 380–1900 mass to charge ratio (m/z)priorto produced in rabbit by injection with the synthetic phosphopolypeptide MS/MS analysis. Raw data were analyzed using Proteome Discoverer™ AGQERFQpSLGVAF (where p stands for phosphorylated residue), coupled software with the Sequest™ algorithm at 15 ppm precursor mass accuracy to keyhole limpet haemocyanin and affinity purified (Sigma). Antibodies and 0.02 Da MS/MS tolerance. The peptide search was performed against and their suppliers were: anti-Rab7 (ab137029, Abcam or D95F2, Cell UniProtKB Homo sapiens reference proteome dataset (release 2012_10) with Signaling), anti-Flag (M2, Sigma or FLA-1, MBL), anti-GFP (JL-8, Clontech a 1% false discovery rate cut-off. The most likely localization of a or 598, MBL), anti-PTEN (138G6, Cell Signaling), anti-Halo (G9281 or phosphorylation site was determined by using the PhosphoRS algorithm G9211, Promega), anti-EEA1 (clone 14, BD Transduction Laboratories), within the Proteome Discoverer software. anti-CD63 (sc5275, Santa Cruz Biotechnology), anti-LAMP1 (H4A3, BD Transduction Laboratories) and anti-RILP (SAB2107831, Sigma). Affinity- Immunoprecipitation and Halo fusion protein pulldown assay purified rabbit antibodies against LRRK1 have been described previously For immunoprecipitation, cells were lysed in RIPA buffer [50 mM Tris-HCl (Hanafusa et al., 2011). Rh–EGF was from Ciliobrevin (Calbiochem) and pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA,

A647–EGF from Invitrogen. 1 mM dithiothreitol, phosphatase inhibitor cocktail 2 (Sigma) and protease Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs228809. doi:10.1242/jcs.228809 inhibitor cocktail (Sigma)], followed by centrifugation at 15,000 g for Author contributions 12 min. The supernatant was added to 50 µl (1.5 mg) of Dynabeads Protein Conceptualization: H.H., K.M.; Methodology: H.H., T.Y., H.I., T.N., K.K., K.S.; G (Invitrogen) with the indicated antibodies (each antibody was used at Validation: H.H.; Formal analysis: H.H., T.Y., H.I., T.N., K.K., K.S.; Investigation: 5 μg/sample) and rotated for 2 h at 4°C. The beads were then washed three H.H., T.Y., H.I., T.N., K.S.; Data curation: H.H.; Writing - original draft: H.H.; Writing - review & editing: N.H., K.M.; Supervision: H.H., N.H., K.K., K.M.; Project times with ice-cold phosphate-buffered saline (PBS) and subjected to administration: H.H., K.M.; Funding acquisition: H.H., K.M. immunoblotting. For Halo-tag pulldown assays, Cos7 cells expressing – – Halo RILP or Halo FYCO1 were lysed in mammalian lysis buffer (G938A, Funding Promega) supplemented with 1 mM dithiothreitol, phosphatase inhibitor This research was supported by grants from Japan Society for the Promotion of cocktail 2 (Sigma) and protease inhibitor cocktail (Promega), followed by Science (JSPS) KAKENHI (17H06001, 18H02612 and 15H02388). centrifugation at 15,000 g for 12 min. The supernatant was added to 100 µl (20% slurry) of Magne-HaloTag beads (Promega), rotated for overnight at Supplementary information 4°C, and then washed three times with ice-cold lysis buffer. HEK293 cells Supplementary information available online at expressing GFP–Rab7 (WT or S72A) with or without GFP– http://jcs.biologists.org/lookup/doi/10.1242/jcs.228809.supplemental LRRK1(Y944F) were lysed in RIPA buffer [50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, 1 mM References dithiothreitol, phosphatase inhibitor cocktail 2 (Sigma) and protease Albeck, J. G., Mills, G. B. and Brugge, J. S. (2013). Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals. Mol. Cell 49, 249-261. inhibitor cocktail (Sigma)] supplemented with 10 mM MgCl2 and doi:10.1016/j.molcel.2012.11.002 0.1 mM GTPγS, followed by centrifugation at 15,000 g for 12 min. The Alegre-Abarrategui, J., Christian, H., Lufino, M. M. P., Mutihac, R., Venda, L. L., supernatants were mixed with Halo–RILP or Halo–FYCO1-bound beads Ansorge, O. and Wade-Martins, R. (2009). LRRK2 regulates autophagic activity and rotated for 2 h at 4°C, and then washed three times with ice-cold lysis and localizes to specific membrane microdomains in a novel human genomic buffer and once with cold PBS. The assays were analyzed by reporter cellular model. Hum. Mol. Genet. 18, 4022-4034. doi:10.1093/hmg/ddp346 immunoblotting with the indicated antibodies. Primary antibodies were Bakker, J., Spits, M., Neefjes, J. and Berlin, I. (2017). The EGFR odyssey-from activation to destruction in space and time. J. Cell Sci. 130, 4087-4096. doi:10. mouse anti-GFP at 1:500 or mouse anti-Halo at 1:1000. 1242/jcs.209197 Biskup, S., Moore, D. J., Celsi, F., Higashi, S., West, A. B., Andrabi, S. A., Phos-tag assay Kurkinen, K., Yu, S. W., Savitt, J. M., Waldvogel, H. J. et al. (2006). Localization For analysis of phosphorylation status of Rab7, 15% Phos-tag precast gels of LRRK2 to membranous and vesicular structures in mammalian brain. Ann. (SuperSep Phos-tag, Wako) were used. After electrophoresis, the Phos-tag Neurol. 60, 557-569. doi:10.1002/ana.21019 Bosgraaf, L. and Van Haastert, P. J. M. (2003). Roc, a Ras/GTPase domain in acrylamide gel was washed three times by gentle shaking in transfer complex proteins. Biochim. Biophys. Acta 1643, 5-10. doi:10.1016/j.bbamcr. buffer (Fast buffer, ATTO) containing 0.01% SDS and 10 mM EDTA for 2003.08.008 10 min and then incubated in transfer buffer containing 0.01% SDS without Bucci, C., Thomsen, P., Nicoziani, P., McCarthy, J., van Deurs, B. and Pfeffer, EDTA for 10 min according to the manufacturer’s protocol. Proteins S. R. (2000). Rab7: a key to lysosome biogenesis. Mol. Biol. Cell 11, 467-480. were transferred to polyvinylidene difluoride (PVDE) membranes and doi:10.1091/mbc.11.2.467 analyzed by immunoblotting with anti-Flag (1:500) or anti-pS72-Rab7 Cantalupo, G., Alifano, P., Roberti, V., Bruni, C. B. and Bucci, C. (2001). Rab- (1:200) antibody. Immunoreactive proteins were visualized and quantified interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes. EMBO J. 20, 683-693. doi:10.1093/emboj/20.4.683 using the FUSION system (VILBER). Ceresa, B. P. and Bahr, S. J. (2006). Rab7 activity affects epidermal growth factor: epidermal growth factor receptor degradation by regulating endocytic trafficking Fluorescence microscopy and image analysis from the late endosome. J. Biol. Chem. 281, 1099-1106. doi:10.1074/jbc. M504175200 For immunofluorescence microscopy, cells were grown on coverslips, Ceresa, B. P. and Peterson, J. L. (2014). Cell and molecular biology of epidermal treated as indicated and then fixed in 4% paraformaldehyde for 15 min at growth factor receptor. Int. Rev. Cell Mol. Biol. 313, 145-178. doi:10.1016/B978-0- 37°C, permeabilized in 0.5% Triton X-100 for 5 min, and incubated with 12-800177-6.00005-0 primary and secondary antibodies. Primary antibodies were rabbit anti-Rab7 Dhekne, H. S., Yanatori, I., Gomez, R. C., Tonelli, F., Diez, F., Schüle, B., Steger, at 1:200, rabbit anti-pS72-Rab7 at 1:250, rabbit anti-Halo at 1:500, anti- M., Alessi, D. R. and Pfeffer, S. R. (2018). A pathway for parkinson’s disease EEA1 at 1:100, anti-CD63 at 1:250, anti-LAMP1 at 1:500. Secondary LRRK2 kinase to block primary cilia and sonic hedgehog signaling in the brain. antibodies were Alexa Fluor 488-, 555- or 647-goat anti-mouse-IgG or anti- eLife 7, e40202. doi:10.7554/eLife.40202 Driskell, O. J., Mironov, A., Allan, V. J. and Woodman, P. G. (2007). Dynein is rabbit-IgG antibodies (Invitrogen). Confocal microscopy was performed required for receptor sorting and the morphogenesis of early endosomes. Nat. Cell using an Olympus FV1000 microscope. Confocal images were captured at Biol. 9, 113-120. doi:10.1038/ncb1525 0.5 µm intervals and z-stacks were processed with FV1000 software. Eden, E. R., White, I. J. and Futter, C. E. (2009). Down-regulation of epidermal Quantification of the colocalization of Rab7 or Halo–RILP with A647–EGF growth factor receptor signalling within multivesicular bodies. Biochem. Soc. was carried out using an ImageJ plug-in (NIH) to generate a binary image of Trans. 37, 173-177. doi:10.1042/BST0370173 the pixels in each image. The ImageJ algorithm generated an automated Firestone, A. J., Weinger, J. S., Maldonado, M., Barlan, K., Langston, L. D., O’Donnell, M., Gelfand, V. I., Kapoor, T. M. and Chen, J. K. (2012). Small- threshold, and colocalization was quantified for pixels whose intensities molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, were higher than the threshold. The number of both A647–EGF-positive 125-129. doi:10.1038/nature10936 vesicles and Rab7- or Halo–RILP-labeled A647–EGF double-positive Futter, C. E. (1996). Multivesicular endosomes containing internalized EGF-EGF vesicles per cell were counted. Data were plotted as the values obtained by receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. dividing Rab7- or Halo–RILP-labeled A647–EGF-positive vesicles by the 132, 1011-1023. doi:10.1083/jcb.132.6.1011 total number of A647–EGF-positive vesicles for each cell. For each series of Goh, L. K. and Sorkin, A. (2013). Endocytosis of receptor tyrosine kinases. Cold Spring Harb. Perspect. Biol. 5, a017459. doi:10.1101/cshperspect.a017459 experiments, the microscope settings were optimized for the brightest Guerra, F. and Bucci, C. (2016). Multiple roles of the small GTPase Rab7. Cells 5, unsaturated images and remained unaltered during analysis. For 34. doi:10.3390/cells5030034 comparisons among samples, we chose cells expressing relatively similar Hanafusa, H., Ishikawa, K., Kedashiro, S., Saigo, T., Iemura, S., Natsume, T., levels of GFP–LRRK1 (Y944F or K1243M mutants), GFP–Rab7, Flag– Komada, M., Shibuya, H., Nara, A. and Matsumoto, K. (2011). Leucine-rich Rab7 (WT, CS, Q67L or Q67L/CS mutants) or Halo–RILP, based on the repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor. Nat. fluorescence intensities of GFP, Flag or Halo, respectively. Commun. 2, e158. doi:10.1038/ncomms1161 Huotari, J. and Helenius, A. (2011). Endosome maturation. EMBO J. 30, 3481-3500. doi:10.1038/emboj.2011.286 Acknowledgements Hutagalung, A. H. and Novick, P. J. (2011). Role of Rab GTPases in membrane traffic We thank M. Fukuda for helpful discussion. and cell physiology. Physiol. Rev. 91, 119-149. doi:10.1152/physrev.00059.2009 Irannejad, R., Tsvetanova, N. G., Lobingier, B. T. and von Zastrow, M. (2015). Competing interests Effects of endocytosis on receptor-mediated signaling. Curr. Opin. Cell Biol. 35,

The authors declare no competing or financial interests. 137-143. doi:10.1016/j.ceb.2015.05.005 Journal of Cell Science

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