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.

Keywords: LRRK2; Lysosome; PKC; Phosphorylation; Rab29.

Fig. 1.

Lysosomal stress leads to LRRK2-independent phosphorylation of Rab29. (A) Phosphorylation of Rab29. upon CQ treatment in a time-dependent manner in HEK293 cells overexpressing GFP–Rab29. The images are representative of n=4 trials. pRab29 and non-pRab29 indicate phosphorylated and nonphosphorylated Rab29, respectively. Error bars indicate s.e.m.; n=4. **P<0.01, ****P<0.0001 [one-way ANOVA followed by Dunnett's test against the control (0 h)]. (B) Phosphorylation of endogenous Rab29 in HEK293 and RAW264.7 cell lines. Representative image of n=3 trials. (C) Phosphorylation of a set of Rab proteins with or without CQ in HEK293 cells. A screening of n=1 trial. (D) Comparison of Rab29 phosphorylation upon CQ treatment or by LRRK2 in HEK293 cells. Representative image of n=4 trials. (E) Quantification of the intensity of the lowermost phosphorylated band in D. Error bars indicate s.e.m.; n=4. ***P<0.001 (one-way ANOVA followed by Dunnett's test against the control). (F) Localization of endogenous Rab29 under CQ-treated conditions in RAW264.7 cells. The arrowhead indicates Rab29 colocalization with LAMP1, a lysosomal marker, on enlarged lysosomes. Scale bars: 10 μm. (G) Quantification of lysosomal localization of Rab29 in each field, as shown in F. Error bars indicate s.e.m.; n=3 fields. ***P<0.001 (unpaired, two-tailed Student's t-test against the control). (H) Biochemical detection of endogenous Rab29 in flow through (FT) and lysosome (Lyso) fractions from HEK293 cells treated with or without CQ. LAMP2, α-tubulin and calnexin were also analyzed as markers of lysosome, cytosol and ER membrane, respectively. Representative image of n=3 trials.

Fig. 2.

Phosphorylation of Rab29 could occur on the lysosomal surface. (A) A scheme of the forced localization technique used in this study. Upon treatment with the heterodimerizer AP21967, FKBP-bound Rab29 is anchored away to FRB-positive compartments in the cell. (B) Phosphorylation of FKBP-Rab29 over time upon forced lysosomal localization by AP21967 in HEK293 cells co-expressing LAMP1–FRB. The images are representative of n=4 trials. Error bars indicate s.e.m.; n=4. *P<0.05, **P<0.01 [one-way ANOVA followed by Dunnett's test against the control (0 h)]. (C,D) Anchoring Rab29 at mitochondria (Fis1) or lysosomes (LAMP1) in HEK293 cells stained with (C) a LAMP1 antibody or (D) MitoTracker Red. Arrowheads indicate Rab29 colocalized with (C) LAMP1 or (D) mitochondria. Scale bars: 10 μm. (E) Phosphorylation of Rab29 upon forced localization to lysosomes (LAMP1) but not to mitochondria (Fis1) in HEK293 cells. Representative image of n=4 trials. (F) Quantification of the phosphorylated bands in E. The percentage of Rab29 phosphorylation (pRab29) was calculated by dividing the intensities of bands indicating pRab29 by the sum of those indicating non-pRab29 and pRab29. n=4. **P<0.01 (one-way ANOVA followed by Tukey's test).

Fig. 3.

Ser185 residue of Rab29 is phosphorylated under lysosomal stress. (A) A representative spectrum of LC-MS/MS indicating phosphorylation at Ser31 or Ser185. (B) A sequence of Rab29 showing the putative phosphorylation sites Ser31 and Ser185. (C) Reduced phosphorylation of Rab29 upon alanine substitution of Ser185, but not Ser31, under CQ treatment in HEK293 cells. Representative image of n=5 trials. (D) Confirmation of Ser185 phosphorylation by using phospho-specific antibodies against HEK293 cell samples. Representative image of n=3 trials. (E) No changes in CQ-induced phosphorylation of Rab29 by inhibition of LRRK2 in HEK293 cells. Representative image of n=3 trials. (F) An AlphaFold2-generated structural image of Rab29. The pink residue pointed to by the pink arrow is Ser185. The orange and green chains indicate switch 1 and 2, respectively. α-Helices are colored in red and β-sheets in blue.

Fig. 4.

Phosphomimetics of Ser185 alleviate CQ-induced lysosomal enlargement. (A) Lysosome morphology and Rab29 localization in HEK293 cells expressing GFP–Rab29 WT or Ser185 mutants at steady state. (B) Lysosome morphology and Rab29 localization in HEK293 cells expressing GFP–Rab29 WT or mutants upon 8 h of CQ treatment. Arrowheads indicate lysosomes with Rab29 accumulation. Scale bars: 10 μm. (C) Statistical analysis of lysosomal size in A and B. Each shape shows the area of the largest lysosome in each cell, obtained by elliptical approximation of each immunocytochemistry image. Only lysosomes from all of the Rab29-positive cells were included in this analysis (92–172 cells in each condition). The mean is shown by a black horizontal bar in each sample. ****P<0.0001 [one-way ANOVA followed by Dunnett's test against the control (wild type, no CQ) sample].

Fig. 5.

PKCs are involved in Ser185 phosphorylation and lysosomal localization of Rab29. (A) In vitro kinase assay using recombinant Rab29 and PKCα or PKCε. (B) Phosphorylation of Rab29 with PMA or Go6983 over time in HEK293 cells. (C) Cells were treated with siRNAs for PKC isozymes that were targeted by both PMA and Go6983, and the phosphorylation of Rab29 upon PMA treatment (24 h) was assessed by Phos-tag or anti-phospho-S185 antibody. Overall PKC activity was additionally monitored by detecting phospho-Ser PKC substrates (pS-PKC substrates). Images in A–C are representative of n=3 trials. (D) Rab29 localization upon PMA treatment in RAW264.7 cells. The arrow indicates colocalization of endogenous Rab29 with LAMP1. Scale bars: 10 μm. (E) Quantification of lysosomal localization of Rab29, as shown in D. Error bars indicate s.e.m.; n=3 fields **P<0.01 (unpaired two-tailed t-test). (F) Lysosomal localization of endogenous Rab29 upon CQ and Go6983 treatment in RAW264.7 cells. Arrowheads indicate enlarged lysosomes with Rab29 accumulation. Scale bars: 10 μm. (G) Quantification of lysosomal localization of Rab29, as shown in F. Error bars indicate s.e.m.; n=3 fields. ****P<0.0001 (one-way ANOVA followed by Tukey's test). (H) Lysosomal localization of endogenous LRRK2 upon CQ and Go6983 treatment in RAW264.7 cells. Arrowheads indicate enlarged lysosomes with Rab29 accumulation. Images in H are representative of three experiments. Scale bars: 10 μm.

Fig. 6.

LRRK2 is also a regulator of Rab29 localization. (A) Rab29 localization upon knockdown of LRRK2 and CQ treatment in RAW264.7 cells. The arrowhead indicates enlarged lysosomes with Rab29 accumulation. (B) Quantification of lysosomal localization of Rab29, as shown in A. Error bars indicate s.e.m.; n=3, 9, 9 fields, respectively. ****P<0.0001 (one-way ANOVA followed by Tukey's test). (C) Rab29 localization upon MLi-2 (a LRRK2 inhibitor) and CQ treatment in RAW264.7 cells. The arrowhead indicates enlarged lysosomes with Rab29 accumulation. (D) Quantification of lysosomal localization of Rab29, as shown in C. Error bars indicate s.e.m.; n=3 fields. ****P<0.0001 (one-way ANOVA followed by Tukey's test). (E) Rab29 localization upon MLi-2 and PMA treatment in RAW264.7 cells. The arrowhead indicates enlarged lysosomes with Rab29 accumulation. (F) Quantification of lysosomal localization of Rab29, as shown in E. Error bars indicate s.e.m.; n=3 fields. **P<0.01 (one-way ANOVA followed by Tukey's test). Scale bars: 10 μm.

Fig. 7.

A model for Rab29 translocation, phosphorylation and their effects under lysosomal stress. Upon stimuli that cause lysosomal overload, (1) Rab29 first associates with the lysosomal membranes, and (2) Rab29 recruits LRRK2 to lysosomal membranes and is phosphorylated by PKCα and/or PCKδ, and LRRK2, which stabilizes Rab29 on lysosomal membranes. Then, (3) Rab29 and LRRK2 on lysosomes induces downstream effects that lead to lysosomal deflation. This figure was created with BioRender.com.