SignificanceLRRK2, a protein kinase related to Parkinson’s disease, is implicated in the maintenance of lysosomes, and a subset of Rab GTPases has been identified as bona fide substrates of LRRK2. Here, we reveal a key stress-responsive pathway composed of Rab7L1, LRRK2, and phosphorylated Rab8/10 involved in lysosomal homeostasis. Lysosomal overload stress induces translocation of Rab7L1 and LRRK2 to lysosomes, where LRRK2 is activated, and stabilizes Rab8 and Rab10 through phosphorylation. The activation of this machinery protects against lysosomal enlargement and upregulates lysosomal secretion through Rab effectors, EHBP1 and EHBP1L1. These findings elucidate a novel regulatory mechanism of Rab GTPases by phosphorylation by LRRK2 in stressed lysosomes, which may also be involved in the pathomechanism of LRRK2-related disorders.

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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.