Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

Abstract

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

The V-ATPase–ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response

“…We also analyzed the involvement of Rab29, a well-known interactor and upstream regulator of LRRK2 ( Eguchi et al, 2018 ; Kuwahara et al, 2020 ; MacLeod et al, 2013 ; Purlyte et al, 2018 ). Similar to LRRK2, endogenous Rab29 was translocated onto lysosomes upon CQ treatment as reported ( Komori et al, 2023 ), whereas knockdown of ATG16L1 did not suppress the lysosomal targeting of Rab29 ( Fig. S4, C and D ).…”

“…Isolation of lysosomes was conducted as described previously ( Eguchi et al, 2018 ; Komori et al, 2023 ). Cells on a 10-cm dish were cultured in DMEM containing 1 mM HEPES-NaOH (pH 7.2) and 10% dextran-coated magnetite (DexoMAG 40; Liquids Research Ltd.) for 24 h, and then chased in normal media for 24 h. Cells were harvested with trypsin, centrifuged at 60 × g for 5 min, washed with ice-cold PBS, lysed in 2 ml of ice-cold Buffer A (1 mM HEPES, 15 mM KCl, 1.5 mM Mg(Ac) 2 , 1 mM DTT, protease/phosphatase inhibitors) with a Dounce homogenizer, and passed through a 23G needle for eight times.…”

An Update on the Interplay between LRRK2, Rab GTPases and Parkinson’s Disease

“…Also known as Rab7L1, Rab29 itself is nominated as a risk factor for PD, encoded in the PARK16 locus [125][126][127]. This GTPase was among the first of all Rabs to have their relationship with LRRK2 uncovered [128,129] and is the Rab that is known to regulate LRRK2 from upstream [16,72,76,87,92,130] in addition to the recently reported Rab12 [53,54] and Rab38 [131]. Rab29 localization at steady state was reported to be at the Golgi, with a small fraction at perinuclear vesicles [132,133].…”

“…Ser106 Perinuclear clustering of lysosomes via increase in RILPL1 binding [86] Activation and recruitment of LRRK2 [53,54] Rab29 Thr71, Ser72 Regulation of trans-Golgi morphology [18] Recruitment of Rab29 itself to stressed lysosomes [87]…”

“…The function of Rab29 has attracted attention not only from its relationship to the Golgi but also to the lysosome, as the small population of Rab29 at perinuclear vesicles was found to be lysosomal. Also, Rab29 has been shown to react to lysosomal stress, localizing itself to lysosomes and also co-recruiting and activating LRRK2 [76,87]. Active Rab29 on lysosomes regulates the size of abnormally inflated lysosomes, which is dependent on LRRK2 kinase activity [76,87].…”