Novel phosphorylation of Rab29 that regulates its localization and 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 was deemed to be responsible for this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCα 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 lysosomal stress responses

“…We also analyzed the involvement of Rab29, a well-known interactor and upstream reglator 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. S4C-D) .…”

“…The copyright holder for this this version posted October 13, 2023. ; https://doi.org/10.1101/2023.10.13.562167 doi: bioRxiv preprint 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. S4C-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.…”