No abstract

Significance Understanding loci nominated by genome-wide association studies (GWASs) is challenging. Here, we show, using the specific example of Parkinson disease, that identification of protein–protein interactions can help determine the most likely candidate for several GWAS loci. This result illustrates a significant general principle that will likely apply across multiple diseases.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domaindomain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.

Mutations in DJ-1, PINK1 (PTEN-induced putative kinase 1) and parkin all cause recessive parkinsonism in humans, but the relationships between these genes are not clearly defined. One event associated with loss of any of these genes is altered mitochondrial function. Recent evidence suggests that turnover of damaged mitochondria by autophagy might be central to the process of recessive parkinsonism. Here, we show that loss of DJ-1 leads to loss of mitochondrial polarization, fragmentation of mitochondria and accumulation of markers of autophagy (LC3 punctae and lipidation) around mitochondria in human dopaminergic cells. These effects are due to endogenous oxidative stress, as antioxidants will reverse all of them. Similar to PINK1 and parkin, DJ-1 also limits mitochondrial fragmentation in response to the mitochondrial toxin rotenone. Furthermore, overexpressed parkin will protect against loss of DJ-1 and, although DJ-1 does not alter PINK1 mitochondrial phenotypes, DJ-1 is still active against rotenone-induced damage in the absence of PINK1. None of the three proteins complex together using size exclusion chromatography. These data suggest that DJ-1 works in parallel to the PINK1/parkin pathway to maintain mitochondrial function in the presence of an oxidative environment.

Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser͞Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial ''kinase-dead'' mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria. mitochondria ͉ PARK6 ͉ Parkinson's disease S everal genes have now been identified that are causally associated with recessive parkinsonism. Mutations in parkin (1) are a relatively frequent cause of parkinsonism with onset before the age of 50, having a varied but generally mild phenotype. DJ-1 mutations are less common than parkin mutations, but the phenotype is broadly similar (2). Given that recessive mutations in either of these two genes cause parkinsonism, it is likely that mutations in either parkin or DJ-1 lead to loss of dopaminergic neurons in the substantia nigra that project to the striatum. Positron-emission tomography demonstrates a loss of dopaminergic function in parkin (e.g., 3-6) and DJ-1 (7, 8) patients, supporting this idea. Therefore, although detailed pathology of these two genetic forms of parkinsonism is not available, there are clear phenotypic overlaps. This conclusion argues that there are relationships between parkin and DJ-1, but the normal functions of the two wild-type proteins are not obviously linked. Parkin is an E3 protein-ubiquitin ligase (9), whereas DJ-1 may have a number of roles (10) but can affect the ability of neurons to survive oxidative stress generated as a result of mitochondrial damage (11).Recently, mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been described that are also associated with recessive parkinsonism. Initially, three pedigrees were described with identified mutations: a G309D point substitution in one family and a truncation mutation (W437X) in two additional families (12). Subsequently, several studies have described co...

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg 2؉ at 2.0-Å resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD. Parkinson's disease (PD) is a common, age-related neurodegenerative disorder for which only symptomatic treatment is available. The etiology of PD is poorly understood, but over the past decade it has become clear that there are rare families with Mendelian inheritance (1). Of the genes that cause PD, dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are numerically the most common and account for an appreciable fraction of apparently sporadic PD (reviewed in ref.2). LRRK2 encodes a large (2,527-aa) multidomain protein originally identified as a unique kinase with leucine-rich repeats. LRRK2 is a member of a superfamily of proteins, named ROCO, that includes at least three other human proteins: leucine-rich repeat kinase 1 (LRRK1), death-associated protein kinase (DAPK1), and malignant fibrous histiocytoma-amplified sequences with leucine-rich tandem repeat-1 (MASL1) (3). A unique feature of all ROCO proteins is a 200-to 250-aa Ras-related GTPase or Ras of complex proteins (ROC) domain, followed by a C-terminal of ROC (COR) domain immediately before the kinase domain.Both LRRK2 and LRRK1 have been shown to be active protein kinases in vitro (4-7), and some mutations are found in the kinase domain. These mutations generally increase kinase activity, although there are some discrepancies in different studies as to whether all mutations increase kinase function (4,6,(8)(9)(10)(11). However, the kinase activity of LRRK2 is required for the ability of the mutant protein to cause neuronal damage, at least in cell culture models (5, 10), suggesting that kinase inhibitors may represent a therapeutic avenue for PD.Although the kinase domain therefore is important in understanding pathogenesis, mutations also are fo...

Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate the how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.