Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology Tetta Fujimoto 1 , Tomoki Kuwahara 1 , Tomoya Eguchi, Maria Sakurai, Tadayuki Komori, Takeshi Iwatsubo * Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, Tokyo,113-0033, Japan article info Article history: Received 1 December 2017 Accepted 5 December 2017 Available online xxx Keywords: Parkinson's disease LRRK2 Rab7L1 Phosphorylation trans-Golgi abstract Mutations in leucine-rich repeat kinase 2 (LRRK2) are the major genetic cause of autosomal-dominantly inherited Parkinson's disease. LRRK2 is implicated in the regulation of intracellular trafficking, neurite outgrowth and PD risk in connection with Rab7L1, a putative interactor of LRRK2. Recently, a subset of Rab GTPases have been reported as substrates of LRRK2. Here we examine the kinase activity of LRRK2 on Rab7L1 in situ in cells. Phos-tag analyses and metabolic labeling assays revealed that LRRK2 readily phosphorylates Golgi-localized wild-type Rab7L1 but not mutant forms that are distributed in the cytoplasm. In vitro assays demonstrated direct phosphorylation of Rab7L1 by LRRK2. Subsequent screening using Rab7L1 mutants harboring alanine-substitution for every single Ser/Thr residue revealed that Ser72 is a major phosphorylation site, which was confirmed by using a phospho-Ser72-specific antibody. Moreover, LRRK2 pathogenic Parkinson mutants altogether markedly enhanced the phos- phorylation at Ser72. The modulation of Ser72 phosphorylation in Rab7L1 resulted in an alteration of the morphology and distribution of the trans-Golgi network. These data collectively support the involvement of Rab7L1 phosphorylation in the LRRK2-mediated cellular and pathogenetic mechanisms. © 2017 Elsevier Inc. All rights reserved. 1. Introduction Parkinson's disease (PD) is a common neurodegenerative dis- order of the adults mainly affecting the extrapyramidal motor systems. The leucine-rich repeat kinase 2 (LRRK2) gene was identi- fied as one of the most common causative genes for autosomal- dominant, late-onset familial PD [1,2]. A number of missense mu- tations in LRRK2 gene have been reported, and a minimum of seven mutations (N1437H, R1441 C/G/H, Y1699C, G2019S, I2020T) are considered as causative and highly penetrant in familial PD [3,4]. Moreover, recent genome-wide association studies (GWAS) in sporadic PD have also identified LRRK2 as one of the strongest risk factors for PD [5]. LRRK2 encodes a large protein harboring multiple domains, e.g., kinase and GTPase (ROC-COR) domains. LRRK2 pos- sesses kinase activity, and the reported substrates include Endo- philin A, ribosomal protein s15 and a subset of Rab GTPases [6e11]. However, the significance of phosphorylation of these substrates remains largely unclear. Accumulating evidence on LRRK2 functions suggest an impor- tant role of LRRK2 in the intracellular trafficking and the mainte- nance of subcellular organs, e.g., Golgi or lysosomes [12]. LRRK2 acts in coordination with multiple functional partners, among which Rab7L1, a member of Rab family of small GTPases, is high- lighted as a key factor. LRRK2 and Rab7L1 bind each other [13,14], coordinately regulating neurite outgrowth and intracellular traf- ficking [13]. In addition, a Rab7L1-LRRK2 genetic pathway functions in diverse organismal and cellular context, ranging from C. elegans neurons to mammalian kidneys, to regulate neurite morphology and lysosome integrity [15]. Importantly, RAB7L1 has been identi- fied as a candidate gene located in a PD risk-associated locus, PARK16 [16], and two genetic variants in LRRK2 and PARK16 impact PD risk in a non-additive manner, suggesting a genetic interaction in humans [13,17]. Rab7L1 protein is mainly localized to or near Golgi apparatus in cells [18]. Although the major role of Rab7L1 is yet to be clarified, prior studies have suggested a role in the modulation of trans-Golgi clustering and integrity [14,19e21]. Wang et al. reported that the impairment of trans-Golgi structure by Rab7L1 knockdown * Corresponding author. Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo Bunkyo-ku, Tokyo, 113-0033, Japan. E-mail address: iwatsubo@m.u-tokyo.ac.jp (T. Iwatsubo). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2017.12.024 0006-291X/© 2017 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2017) 1e8 Please cite this article in press as: T. Fujimoto, et al., Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans- Golgi morphology, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.024