Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Kluss, Jillian H.; Bonet-Ponce, Luis; Lewis, Patrick A.; Cookson, Mark

doi:10.1016/j.nbd.2022.105769

Cited by 27 publications

(23 citation statements)

References 48 publications

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“…One study using a mitochondrially tagged Rab29 protein showed that directing LRRK2 to the mitochondrial membrane is sufficient to induce its activation and downstream Rab10 phosphorylation ( 18 ), suggesting that specific membrane identity is unimportant in LRRK2 activation. We have recently confirmed this finding at other membranes, in which directing LRRK2 to early, late, and recycling endosomes, plasma membrane, Golgi, and lysosomes was sufficient to activate LRRK2 kinase ( 19 ). However, how Rab phosphorylation patterns vary across different sub-cellular compartments remains to be defined.…”

supporting

confidence: 54%

“…Briefly, in the presence of rapamycin, these domains form a heterodimer, and rapidly and irreversibly direct a target protein to a target membrane ( 21 ). We therefore tagged LRRK2 with the FKBP sequence (3xFLAG-FKBP-LRRK2) and fused the FRB sequence to the lysosomal and early endosomal membrane markers lysosomal-associated membrane protein 1 (LAMP1) and Rab5, respectively, which we have previously characterized ( 19 ). Second, we cloned two chimera-LRRK2 constructs by tagging the N terminus of LRRK2 with a 39–amino acid (aa) transmembrane sequence from LAMTOR1 for lysosomal targeting and membrane-associated double zinc finger and coiled-coil domains FYVE-CC2 from hepatocyte growth factor (HRS) protein for early endosome targeting ( 22 , 23 ).…”

Section: Resultsmentioning

confidence: 99%

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Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes

Kluss

Beilina

Williamson

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Genetic variation at the leucine-rich repeat kinase 2 ( LRRK2 ) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2 + lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

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supporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes

Kluss

Beilina

Williamson

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Both the anterograde motor kinesin-1 and the retrograde motor cytoplasmic dynein (in complex with dynactin) are bound to AVs, but adaptor proteins inhibit kinesin and promote dynein activity in WT neurons (Cason et al, 2021; Cheng et al, 2015; Fu et al, 2014; Maday et al, 2012). In our previous work, we found that hyperactive LRRK2 recruits the motor adaptor JIP4, which specifically binds to LRRK2-phosphorylated RAB proteins, to the AV membrane (Boecker et al, 2021; Bonet-Ponce et al, 2020; Kluss et al, 2022; Waschbüsch et al, 2020). This results in abnormal recruitment and activation of kinesin that disrupts processive retrograde AV transport (Boecker et al, 2021).…”

Section: Resultsmentioning

confidence: 98%

“…Hyperactive LRRK2 mutations induce increased phosphorylation of RABs, altering their interactions with downstream binding partners. These binding partners include the motor adaptor proteins JNK-interacting protein 3 and 4 (JIP3/4), which selectively interact with LRRK2-phosphorylated RAB proteins and control activation of the molecular motors kinesin and dynein (Bonet-Ponce et al, 2020; Kluss et al, 2022; Waschbüsch et al, 2020). Altered binding to effector proteins induced by LRRK2-mediated phosphorylation of RABs is predicted to alter RAB-dependent intracellular vesicle transport.…”

Section: Introductionmentioning

confidence: 99%

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Dou

Smith

Evans

et al. 2022

Preprint

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Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We found that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin motors. In iPSC-derived human neurons, knock-in of the strongly-hyperactive LRRK2-p.R1441H mutation caused striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knock-out of the opposing Protein Phosphatase 1H (PPM1H) phenocopied the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuated transport defects in both p.R1441H knock-in and PPM1H knock-out neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

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“…Consensus of past literature is that LRRK2 phosphorylates Rab29 (Thr71 and Ser72 10,12 ), and Rab29 recruits and activates LRRK2 (either at the Golgi or lysosomes 19,21,38 ). The exact mechanism of LRRK2 activation also is unclear, but possibly mediated by heteromultimer formation with Rab29 and/or by membrane association of LRRK2 25,39 . Our data suggest that the regulation of the localization of Rab29 and LRRK2 is intertwined, both needing each other to be stably localized on membranes.…”

Section: Discussionmentioning

confidence: 99%

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

Komori

Kuwahara

Fujimoto

et al. 2022

Preprint

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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 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.

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Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Cited by 27 publications

References 48 publications

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

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Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Cited by 27 publications

References 48 publications

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 + lysosomes

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 + lysosomes

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

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Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 ⁺ lysosomes