Biology subjects

Alessi, D. R.

Publications and source records attributed to Alessi, D. R..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Genome-wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence overall phosphoRab levels in a manner distinct from Rab29 activation of LRRK2. Our data support a model in which Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

biochemistry↗

bioRxiv2022

Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content

The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signalling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified novel ones. Metabolite profiling established the first known human Golgi metabolome and revealed the selective enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease states. SignificanceThe Golgi is central to protein and lipid processing. It senses and responds to diverse cell states to allow trafficking of macromolecules based on cellular demands. Traditional techniques for purifying the Golgi shaped our understanding of its functions, however such methods are too slow to preserve the labile Golgi metabolome and transient protein interactions. Here, we overcome this issue through the development of a method for the rapid capture of intact Golgi from human cells using organelle-specific immunoprecipitation (Golgi-IP). Using high resolution mass spectrometry, we demonstrate that our approach allows the unbiased characterization of the Golgi proteome, metabolome and lipidome. Thus, we believe that the Golgi-IP will be useful for the study of the Golgi in health and disease states.

cell biology↗

bioRxiv2022

PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain

Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinsons disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKC unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain C-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ~3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the aC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

biochemistry↗

bioRxiv2022

Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration

The most common genetic mutation found in familial and sporadic amyotrophic lateral sclerosis (ALS), as well as fronto-temporal dementia (FTD), is a repeat expansion in the C9orf72 gene. C9orf72 is highly expressed in human myeloid cells, and although neuroinflammation and microglial pathology are widely found in ALS/FTD, the underlying mechanisms are poorly understood. Here, using human induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harbouring C9orf72 mutation (mC9-MG) together with gene-corrected isogenic controls (isoC9-MG) and C9ORF72 knock-out hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated inflammatory response upon stimulation with lipopolysaccharide, driven by sustained activation of NLRP3 inflammasome and NF-{kappa}B signalling. Analysis of the hiPSC-MG C9ORF72 interactome revealed an association of C9ORF72 with key regulators of autophagy, a process involved in the homeostatic regulation of the innate immune response. We found impaired initiation of autophagy in C9KO-MG and mC9-MG. Furthermore, through motor neuron-microglial (MN-MG) co-culture studies, we identified that autophagy deficit in mC9-MG led to increased vulnerability of C9 MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated the sustained activation of NLRP3 inflammasome and NF-{kappa}B signalling, reversed the phagocytic deficit found in mC9-MG and also reduced MN death in MN-MG co-cultures. We validated these findings in blood-derived macrophages from people with C9orf72 mutation. Our results reveal an important role for C9ORF72 in regulating microglial immune homeostasis and identify dysregulation in human myeloid cells as a contributor to neurodegeneration in ALS/FTD. TeaserDisrupted autophagy led immune activation in microglia results in enhanced motor neuronal death in C9orf72-ALS.

neuroscience↗

bioRxiv2022

Structural basis of human LRRK2 membrane recruitment and activation

Mutations in LRRK2 are the most common genetic cause of late-onset Parkinsons disease (PD). LRRK2 encodes the leucine-rich repeat kinase 2 (LRRK2), whose kinase activity is regulated by Rab29, a membrane-anchored GTPase. However, molecular mechanisms underlying Rab29-dependent recruitment and activation of LRRK2 remain unclear. Here we report cryo-EM structures of LRRK2-Rab29 complexes in three oligomeric states, illustrating snapshots of key steps during LRRK2 membrane recruitment and activation. Rab29 binds to the ARM domain of LRRK2, and disruption at the interface abrogates LRRK2 kinase activity. Activation of LRRK2 is underpinned by the formation of an unexpected Rab29-induced super-assembly containing two central kinase-active and two peripheral kinase-inactive LRRK2 protomers. Central protomers undergo pronounced oligomerization-associated rearrangements and adopt an active conformation. Our work reveals the structural mechanism for LRRK2s spatial regulation controlled by Rab GTPases, provides mechanistic insights into pathogenic mutations and identifies new opportunities to design LRRK2 inhibitors for PD treatment.

biophysics↗

bioRxiv2022

A Feed-forward Pathway Drives LRRK2 kinaseMembrane Recruitment and Apparent Activation

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinsons disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed "Site #1", can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed "Site #2", that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

biochemistry↗

bioRxiv2022

Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding

Mutations enhancing the kinase activity of LRRK2 cause Parkinsons disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

neuroscience↗

bioRxiv2022

A mitochondrial blood-based patient stratification candidate biomarker for Parkinson's disease

Parkinsons disease (PD) is the most common neurodegenerative movement disorder and neuroprotective interventions remain elusive. High throughput biomarkers aimed to stratify patients based on shared etiology is one critical path to the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific mitochondrial DNA (mtDNA) damage accumulation in neuronal and in vivo PD models, as well as human PD postmortem brain tissue. In this study, to investigate mtDNA damage as a potential blood biomarker for PD, we describe a novel Mito DNADX assay that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, compatible with assessing large cohorts of patient samples. We found that levels of mtDNA damage were increased in blood derived from early-stage idiopathic PD patients or those harboring the pathogenic LRRK2 G2019S mutation compared to age-matched healthy controls. Given that increased mtDNA damage was also found in non-manifesting LRRK2 mutation carriers, mtDNA damage may begin to accumulate prior to a clinical PD diagnosis. LRRK2 kinase inhibition mitigated mtDNA damage in idiopathic PD models and patient-derived cells. The latter observations further substantiate a mechanistic role for wild-type LRRK2 kinase activity in idiopathic PD and support mtDNA damage reversal as a suitable approach to slow PD-related pathology. In light of recent advances in the field of precision medicine, the analysis of mtDNA damage as a blood-based patient stratification biomarker should be included in future clinical trials. One Sentence SummaryBlood test identifies Parkinsons patients most likely to respond to mitochondria-targeted therapeutics facilitating a precision medicine approach.

neuroscience↗

bioRxiv2021

Impact of Type II LRRK2 inhibitors on signalling and mitophagy

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinsons disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

neuroscience↗

bioRxiv2021

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Previously, we showed that cholinergic interneurons of the dorsal striatum lose cilia in mice harboring the Parkinsons disease associated, kinase activating, R1441C LRRK2 mutation (Dhekne et al., 2018). Here we show that this phenotype is also seen in two mouse strains carrying the most common human G2019S LRRK2 mutation. Heterozygous loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases (Berndsen et al., 2019) yields the same cilia loss phenotype, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. In addition, astrocytes throughout the striatum show a ciliation defect in LRRK2 and PPM1H-/+ mutant models. Hedgehog signaling requires cilia, and loss of cilia correlates here with a loss in induction of Hedgehog signaling as monitored by in situ hybridization of Gli1 transcripts. These data support a model in which LRRK2 and PPM1H mutant mice struggle to receive and respond to critical Hedgehog signals in the nigral-striatal pathway.

cell biology↗

bioRxiv2021

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

LRRK2 serine/threonine kinase is associated with inherited Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity toward substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110-residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

biochemistry↗

bioRxiv2020

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinsons disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinsons mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

biochemistry↗

bioRxiv2020

Development of a multiplexed targeted mass spectrometry assay for LRRK2 phosphorylated Rabs and Ser910/Ser935 biomarker sites

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinsons pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinsons patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

neuroscience↗

bioRxiv2020

Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase

The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsBTBBric-a-brac, Tramtrack, and Broad complexCCTConserved C-terminalCRL3Cullin3-dependent RING E3 ligaseDMEMDulbecco’s modified Eagle’s mediumDTTDithiothreitolFBSfetal bovine serumGSTGlutathione S-transferaseHCDHigher-energy collisional dissociationHEK293Thuman embryonic kidney 293 cell line expressing the SV40 large T antigenKCCK+/Cl− co-transportersIPTGIsopropyl β-D-1-thiogalactopyranosideKLHL3Kelch-like protein 3LC/MSLiquid chromatography-mass spectrometryN[K]CCNa+/Cl− ion co-transportersNKCC2Na+/K+/2Cl− co-transporter 2OSR1oxidative stress-responsive kinase 1PDBProtein DatabankPHAIIpseudohypoaldosteronism type IIRINGreally interesting new geneSPAKSPS1-related proline/alanine-rich kinaseTCEPtris(2-carboxyethyl)phosphineTEVtobacco etch virusWNKwith no lysine (K) kinaseView Full Text

molecular biology↗

bioRxiv2020

Rab29 knock-out or transgenic overexpression does not impact basal LRRK2 activity in wildtype and pathogenic mouse and cell line models

Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinsons disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinsons, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on assessment of Rab10 and Rab12 phosphorylation, in wildtype LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that monovalent cation ionophore antibiotics nigericin and monensin enhance LRRK2-mediated Rab10 and Rab12 phosphorylation 4 to 9-fold, in a manner that is independent from Rab29. Moderate stimulation of Rab10 and Rab12 induced by lysosome stressors chloroquine and LLOMe was also not regulated by Rab29. Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.

neuroscience↗

bioRxiv2019

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinsons disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. However, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. We developed an ultra-sensitive targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in human neutrophils before and after LRRK2 inhibition. Compared to healthy controls, neutrophils of LRRK2 G2019S and VPS35 D620N carriers robustly display 1.4-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

biochemistry↗

bioRxiv2019

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is upregulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including 4 endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are poorly phosphorylated by Akt as they possess an n+1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

biochemistry↗

bioRxiv2019

Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

Rab8a GTPase is associated with the dynamic regulation of membrane protrusions in polarized cells. Rab8a is one of several Rab-family GTPases that are substrates of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase that is linked to inherited Parkinsons disease. Rab8a is phosphorylated at T72 (pT72) in its switch 2 helix and the post-translational modification facilitates phospho-Rab8a (pRab8a) interactions with RILPL2, which subsequently regulates ciliogenesis. Here we report the crystal structure of pRab8a in complex with the phospho-Rab binding domain of RILPL2. The complex is a heterotetramer with RILPL2 forming a central -helical dimer that bridges two pRab8a molecules. The N-termini of the -helices cross over to form an X-shaped cap (X-cap) that enables electrostatic interactions between Arg residues from RILPL2 and the phosphate moiety from pT72. RILPL2 residues in the X-cap that are critical for pRab8a binding are conserved in the RILP family of effector proteins. We find that JIP3 and JIP4 also interact specifically with LRRK2-phosphorylated Rab10, suggesting a general mode of recognition for phosphorylated Rab GTPases by phospho-specific effectors.

biochemistry↗