Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of autosomal dominant Parkinson's disease (PD). LRRK2, a member of the ROCO protein family, contains both Ras GTPase-like (Roc) and kinase (MAPKKK) domains, as well as other functional motifs. Here, we have identified LRRK2 as the first mammalian ROCO protein that is an authentic and functional GTPase, defined by the ability to bind GTP and undergo intrinsic GTP hydrolysis. Furthermore, the Roc domain is sufficient for this native GTPase activity and binds and hydrolyzes GTP indistinguishably from the Ras-related small GTPase, Rac1. The PD-associated mutation, R1441C, located within the Roc domain, leads to an increase in LRRK2 kinase activity and a decrease in the rate of GTP hydrolysis, compared to the wild-type protein, in an in vitro assay. This finding suggests that the R1441C mutation may help stabilize an activated state of LRRK2. Additionally, LRRK2 mediated phosphorylation is stimulated upon binding of non-hydrolyzable GTP analogs, suggesting that LRRK2 is a MAPKKK activated intramolecularly by its own GTPase. Since GTPases and MAPKKKs are upstream regulators of multiple signal transduction cascades, LRRK2 may play a central role in integrating pathways involved in neuronal cell signaling and the pathogenesis of PD.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson's disease (PD). While this multi-domain protein has been shown to have both GTPase and kinase activities through the Roc and MAPKKK domains, respectively, the proteinprotein interactions and pathways involved in LRRK2-mediated signaling remain elusive. Utilizing a combination of protein pull-down assays, mass spectrometry, Western blotting and immunofluorescence microscopy, this study identifies and describes the interaction between LRRK2 and microtubules. The Roc or GTPase-like domain of LRRK2 is sufficient for interaction with α/β-tubulin heterodimers. This interaction occurs in a guanine-nucleotide independent manner, suggesting that tubulin may not be an effector of the LRRK2 GTPase domain. The R1441C pathogenic mutation, located within the Roc domain, retains interaction with α/β-tubulin heterodimers, suggesting that disruption of this interaction is not likely the mechanism whereby the R1441C mutation leads to disease. At a subcellular level, endogenous LRRK2 protein was found to colocalize with α/β-tubulin in primary hippocampal neurons. These findings are significant because they link LRRK2 with microtubules, a structural component of the cell that is critically involved in the pathogenesis of several neurodegenerative diseases, including PD.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder with a prevalence of more than 1% after the age of 65 years. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have recently been linked to autosomal dominant, late-onset PD that is clinically indistinguishable from typical, idiopathic disease. LRRK2 is a multi-domain protein containing several protein interaction motifs as well as dual enzymatic domains of GTPase and protein kinase activities. Disease-associated mutations are found throughout the multi-domain structure of the protein. LRRK2, however, is unique among the PD-causing genes because a missense mutation, G2019S, is a frequent determinant of not only familial, but also of sporadic PD. Thus, LRRK2 has emerged as a promising therapeutic target for combating PD. This article reviews the current state of knowledge regarding the domain structure, amino acid substitutions, and potential functional roles of LRRK2.

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation.