Endogenous Rab38 regulates LRRK2’s membrane recruitment and substrate Rab phosphorylation in melanocytes
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ABSTRACT:
SUBMITTER: Unapanta A
PROVIDER: S-EPMC10551901 | biostudies-literature | 2023 Aug
REPOSITORIES: biostudies-literature
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Project description:Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway.
| S-EPMC9665168 | biostudies-literature
Project description:LRRK2 kinase mutations cause familial Parkinson's disease and increased phosphorylation of a subset of Rab GTPases. Rab29 recruits LRRK2 to the trans-Golgi and activates it there, yet some of LRRK2's major Rab substrates are not on the Golgi. We sought to characterize the cell biology of LRRK2 activation. Unlike other Rab family members, we show that Rab29 binds nucleotide weakly, is poorly prenylated, and is not bound to GDI in the cytosol; nevertheless, Rab29 only activates LRRK2 when it is membrane bound and GTP bound. Mitochondrially anchored, GTP-bound Rab29 is both a LRRK2 substrate and activator, and it drives accumulation of active LRRK2 and phosphorylated Rab10 on mitochondria. Importantly, mitochondrially anchored LRRK2 is much less capable of phosphorylating plasma membrane-anchored Rab10 than soluble LRRK2. These data support a model in which LRRK2 associates with and dissociates from distinct membrane compartments to phosphorylate Rab substrates; if anchored, LRRK2 can modify misdelivered Rab substrates that then become trapped there because GDI cannot retrieve them.
| S-EPMC6891090 | biostudies-literature
Project description:BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.MethodsIn vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.ResultsOur screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.ConclusionsHere we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.
| S-EPMC5811984 | biostudies-literature
Project description:Pathogenic missense mutations in the leucine-rich repeat kinase 2 gene, encoding LRRK2, results in the upregulation of Rab10 and Rab12 phosphorylation in different cells and tissues. Here, we evaluate levels of the LRRK2 kinase substrates pT73-Rab10 and pS106-Rab12 proteins in rat brain tissues from different genetic backgrounds. Whereas lines of Sprague Dawley rats have equivalent levels of pT73-Rab10 and pS106-Rab12 similar to Lrrk2 knockout rats, Long-Evans rats have levels of pT73-Rab10 and pS106-Rab12 comparable to G2019S-LRRK2 BAC transgenic rats. Strong LRRK2 kinase inhibitors are ineffective at reducing pT73-Rab10 and pS106-Rab12 levels in the Sprague Dawley rats, but potently reduce pT73-Rab10 and pS106-Rab12 levels in Long-Evans rats. Oral administration of the PFE-360 LRRK2 kinase inhibitor fails to provide neuroprotection from dopaminergic neurodegeneration caused by rAAV2/1-mediated overexpression of A53T-αsynuclein in Sprague Dawley rats. These results highlight substantial differences in LRRK2-mediated Rab10 and Rab12 phosphorylation in commonly utilized rat genetic backgrounds and suggest LRRK2 may not play a central role in Rab phosphorylation or mutant αsynuclein toxicity in Sprague Dawley rats.
| S-EPMC8006086 | biostudies-literature
Project description:Melanosomes are melanin-containing organelles in melanocytes, and they are responsible for skin and hair pigmentation in mammals. The intracellular distribution of melanosomes is mainly determined by the balance between their anterograde transport on actin filaments and retrograde transport on microtubules. Although we have shown previously that melanoregulin and Rab36 serve as cargo receptors on melanosomes for retrograde transport, their knockdown does not completely inhibit retrograde melanosome transport, suggesting the existence of an additional cargo receptor(s) in melanocytes. In this study, we investigated the possible involvement of an atypical large Rab, Rab44, which also contains EF-hand domains and a coiled-coil domain, in retrograde melanosome transport in mouse melanocytes (Rab27A-deficient melan-ash cells). Our results showed that Rab44 localizes on mature melanosomes through lipidation of its C-terminal Rab-like GTPase domain, and that its knockdown results in suppression of retrograde melanosome transport. In addition, our biochemical analysis indicated that Rab44 interacts with the dynein-dynactin motor complex via its coiled-coil domain-containing middle region. Since simultaneous depletion of Rab44, melanoregulin, and Rab36 resulted in almost complete inhibition of retrograde melanosome transport, we propose that Rab44 is the third cargo receptor. We also showed that the N-terminal region of Rab44, which contains EF-hand domains, is required for both retrograde melanosome transport and its Ca2+-modulated activities. Our findings indicated that Rab44 is a third melanosomal cargo receptor, and that, unlike other cargo receptors previously described, its transport function is regulated by Ca2+.
| S-EPMC9586991 | biostudies-literature
Project description:Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.
| S-EPMC7696345 | biostudies-literature
Project description:We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.
| S-EPMC5695910 | biostudies-literature
Project description:Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical-shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C-terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.
| S-EPMC6032855 | biostudies-literature
Project description:Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.
| S-EPMC4769169 | biostudies-literature
Project description:Rab GTPases are master regulators of membrane trafficking in eukaryotic cells. Phosphorylation of Rab GTPases was characterized in the 1990s and there have been intermittent reports of its relevance to Rab functions. Phosphorylation as a regulatory mechanism has gained prominence through the identification of Rabs as physiological substrates of leucine-rich repeat kinase 2 (LRRK2). LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson disease. In recent years, numerous kinases and their associated signaling pathways have been identified that lead to phosphorylation of Rabs. These emerging studies suggest that serine/threonine and tyrosine phosphorylation of Rabs may be a widespread and under-appreciated mechanism for controlling their membrane trafficking functions. Here we survey current knowledge of Rab phosphorylation and discuss models for how this post-translational mechanism exerts control of membrane trafficking.
| S-EPMC7756361 | biostudies-literature