Roc Domain Research Articles

Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations.

Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.

Revisiting the Roco G-protein cycle.

Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.

Structural biology of the LRRK2 GTPase and kinase domains: implications for regulation.

Human leucine rich repeat kinase 2 (LRRK2) belongs to the Roco family of proteins, which are characterized by the presence of a Ras-like G-domain (Roc), a C-terminal of Roc domain (COR), and a kinase domain. Mutations in LRRK2 have been found to be thus far the most frequent cause of late-onset Parkinson’s disease (PD). Several of the pathogenic mutations in LRRK2 result in decreased GTPase activity and enhanced kinase activity, suggesting a possible PD-related gain of abnormal function. Important progress in the structural understanding of LRRK2 has come from our work with related Roco proteins from lower organisms. Atomic structures of Roco proteins from prokaryotes revealed that Roco proteins belong to the GAD class of molecular switches (G proteins activated by nucleotide dependent dimerization). As in LRRK2, PD-analogous mutations in Roco proteins from bacteria decrease the GTPase reaction. Studies with Roco proteins from the model organism Dictyostelium discoideum revealed that PD mutants have different effects and most importantly they explained the G2019S-related increased LRRK2 kinase activity. Furthermore, the structure of Dictyostelium Roco4 kinase in complex with the LRRK2 inhibitor H1152 showed that Roco4 and other Roco family proteins can be important for the optimization of the current, and identification of new, LRRK2 kinase inhibitors. In this review we highlight the recent progress in structural and biochemical characterization of Roco proteins and discuss its implication for the understanding of the complex regulatory mechanism of LRRK2.

Prediction of the Repeat Domain Structures and Impact of Parkinsonism-Associated Variations on Structure and Function of all Functional Domains of Leucine-Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.

A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

Evidence that the LRRK2 ROC domain Parkinson's disease‐associated mutants A1442P and R1441C exhibit increased intracellular degradation

Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T.

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Differential Effects of Familial Parkinson Mutations in LRRK2 Revealed by a Systematic Analysis of Autophosphorylation

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified in pedigrees of autosomal-dominant familial Parkinson's disease (PARK8). It has been shown that the kinase activity of LRRK2 is required for its neuronal toxicity, although how familial Parkinson mutations affect the function of LRRK2 has not been well characterized. In the present study, we systematically characterized the autophosphorylation of LRRK2 by phosphopeptide mapping and identified Thr1348, Thr1349, and Thr1357 as the major autophosphorylation sites. We found that the autophosphorylation at Thr1357 is downregulated by the Y1699C mutation, possibly through a conformational alteration of the ROC domain. We also found that I2020T mutant LRRK2 undergoes excessive autophosphorylation in cell lysates in vitro at a low concentration of ATP. These results highlight the differential effects of familial mutations in LRRK2 on its conformation and enzymatic properties.

Electron Microscopy Structure of Dimeric LRRK2 Reveals a Structural and Regulatory Role of the COR Domain

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) constitute the most common genetic cause of Parkinson disease (PD). LRRK2 is a 286-kDa multidomain protein containing several protein-protein interaction domains, including armadillo, leucine-rich and ankyrin repeats, as well as a Ras Of Complex proteins GTPase (ROC) and a kinase joined by a C-terminal Of ROC (COR) domain. PD-linked mutations are clustered in the catalytic core of LRRK2 suggesting that altered GTPase and kinase activities may be implicated in pathogenesis.

GTPase activity regulates kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2

Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1.

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.

Genetic analysis of LRRK2 functional domains in Brazilian patients with Parkinson’s disease

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been associated with Parkinson's disease (PD), and the majority of the pathogenic variants are located in the ROC and MAPKKK domains. Exons 29-31 and 38-44 (ROC and MAPKKK domains) were sequenced in 204 patients with PD, mostly Brazilian. We identified four polymorphisms, a novel silent variant p.R1398R and four substitutions: p.T1410M, p.G2019S, p.Y2189C and the novel variant p.C2139S. The most prevalent mutation was the p.G2019S (2.4%). We consider that the p.T1410M and the p.Y2189C variants are probably polymorphisms and that the p.C2139S mutation is potentially pathogenic.

Leucine-rich repeat kinase 2-linked Parkinson's disease: clinical and molecular findings.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of sporadic and familial late onset Parkinson’s disease (PD). The G2019S common mutation has been identified about 1% of sporadic cases and 4–7% of familial cases. Over 50 variants have since been identified in LRRK2, and at least 7 of these are confirmed to be pathogenic. In addition to pathogenic mutations, several common polymorphisms in the LRRK2 gene (G2385R and R1628P) have been identified that may explain up to 10% of sporadic PD in Asian populations. LRRK2 is a large complex multidomain protein with 2,527-amino-acid and the molecular weight is 286 kDa. LRRK2 multidomain protein consists of a catalytic core domain, kinase domain and a number of putative protein-protein interaction domains. LRRK2 mutations found in PD families, including the G2019S and I2020T mutations show increased intrinsic kinase activity, when assessed with myelin basic protein as substrate. The modification of LRRK2 GTPase and kinase activity affecting residues in the ROC, COR and mitogen-activated protein kinase kinase kinases domains is believed to lead to neuronal cell death, but the pathways involved remain unclear. A number of in vivo models in C. elegans, D. melanogaster and mice have been developed to study the patho/physiological function of LRRK2. Based on current literature, a toxic gain of function in LRRK2 kinase activity is a possible pathophysiologic mechanism and thus inhibition of kinase activity in experimental models offers a potential therapeutic strategy for LRRK2-linked PD.

Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease

Genealogical investigation of a large Norwegian family (F04) with autosomal dominant parkinsonism has identified 18 affected family members over four generations. Genetic studies have revealed a novel pathogenic LRRK2 mutation c.4309 A>C (p.Asn1437His) that co-segregates with disease manifestation (LOD = 3.15, θ = 0). Affected carriers have an early age at onset (48 ± 7.7 SD years) and are clinically asymmetric and levodopa responsive. The variant was absent in 623 Norwegian control subjects. Further screening of patients from the same population identified one additional affected carrier (1 of 692) with familial parkinsonism who shares the same haplotype. The mutation is located within the Roc domain of the protein and enhances GTP-binding and kinase activity, further implicating these activities as the mechanisms that underlie LRRK2-linked parkinsonism.

Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity associate with familial forms of Parkinson disease (PD). As phosphorylation determines the functional state of most protein kinases, we systematically mapped LRRK2 phosphorylation sites by mass spectrometry. Our analysis revealed a high degree of constitutive phosphorylation in a narrow serine-rich region preceding the LRR-domain. Allowing de novo autophosphorylation of purified LRRK2 in an in vitro autokinase assay prior to mass spectrometric analysis, we discovered multiple sites of autophosphorylation. Solely serine and threonine residues were found phosphorylated suggesting LRRK2 as a true serine threonine kinase. Autophosphorylation mainly targets the ROC GTPase domain and its clustering around the GTP binding pocket of ROC suggests cross-regulatory activity between kinase and Roc domain. In conclusion, the phosphoprotein LRRK2 functions as an autocatalytically active serine threonine kinase. Clustering of phosphosites within two discrete domains suggest that phosphorylation may regulate its biological functions in a yet unknown fashion.

The Small GTPase Activity of the ROC Domain from LRRK2, a Parkinsons Disease Related Protein

Mutations in the LRRK2 gene have been implicated in the pathogenesis of Parkinson's disease. This work provides biochemical evidence that the ROC domain of LRRK2 functions as a small GTPase, and the Parkinson's disease-associated mutants do not appear to have reduced GTP hydrolysis activities.

Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.

Identification of the Autophosphorylation Sites of LRRK2

Parkinson's disease (PD) is a major adult-onset neurodegenerative disorder affecting the extrapyramidal motor system. A subset of patients develop PD as an autosomal dominant trait, of which PARK8 caused by mutations in the leucine-rich repeat kinase 2 (LRRK2) gene is highlighted because of its high frequency and clinicopathological similarity to sporadic PD. Previous studies have suggested that overactivation of LRRK2 caused by missense mutations leads to neuronal toxicity in PARK8, although the regulatory mechanism that governs the kinase activity of LRRK2 remains unknown. In this study, we expressed the carboxyl-half fragments of LRRK2 (DeltaN-LRRK2) that harbors the kinase as well as the ras-like (ROC) domains in Sf9 cells, subjected them to in vitro phosphorylation reaction, and analyzed the autophosphorylation by matrix assisted laser desorption/ionization- time of flight (MALDI-TOF) mass spectrometer. We identified Ser1403, Thr1404, Thr1410, Thr1491 located within the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, as the autophosphorylation sites. Substitution of Thr1967, an autophosphorylation site located within the kinase domain, to Ala caused a significant decrease in the kinase activity, implicating Thr1967 in the kinase activity of LRRK2. Phosphospecific antibodies to the autophosphorylation sites specifically recognized full-length LRRK2 subjected to in vitro phosphorylation reaction, indicating that the autophosphorylation takes place in holoproteins. Further analysis of autophosphorylation will clarify the mechanism of activation of LRRK2, as well as the pathomechanism of PD in relation to overactivation of LRRK2.

The R1441C mutation alters the folding properties of the ROC domain of LRRK2

LRRK2 is a 250 kDa multidomain protein, mutations in which cause familial Parkinson's disease. Previously, we have demonstrated that the R1441C mutation in the ROC domain decreases GTPase activity. Here we show that the R1441C alters the folding properties of the ROC domain, lowering its thermodynamic stability. Similar to small GTPases, binding of different guanosine nucleotides alters the stability of the ROC domain, suggesting that there is an alteration in conformation dependent on GDP or GTP occupying the active site. GTP/GDP bound state also alters the self-interaction of the ROC domain, accentuating the impact of the R1441C mutation on this property. These data suggest a mechanism whereby the R1441C mutation can reduce the GTPase activity of LRRK2, and highlights the possibility of targeting the stability of the ROC domain as a therapeutic avenue in LRRK2 disease.

Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways

Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2–DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2–DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2–DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2–DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.