© 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 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs228809. doi:10.1242/jcs.228809 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,