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 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...

The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys 106 in human DJ-1). Mutation of Cys 106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys 106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu 18 ) in human DJ-1 that alter the oxidative propensity of Cys 106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys 106 to be oxidized to Cys 106 -sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys 106 -sulfinic acid is a key modification that regulates the protective function of DJ-1.Reactive cysteine residues are susceptible to a variety of covalent modifications that are increasingly recognized as a major means of regulating the activities of many proteins (1). Cysteine forms three different species by the direct addition of oxygen; cysteine-sulfenic (-SOH), -sulfinic (-SO 2 H), and -sulfonic (-SO 3 H) acid. Because cysteine can be oxidized to three distinct species, each with different structural and chemical properties, cysteine oxidation is a versatile way for reactive oxygen species (ROS) 4 to alter the activity of a protein.Of the three oxidation products of cysteine, only cysteine-sulfenic acid is readily reduced to the thiol under physiological conditions. However, enzymes that catalyze the ATP-dependent reduction of overoxidized peroxiredoxins containing cysteine-sulfinic acid to cysteine have been discovered and characterized (2, 3). With reversibility comes the potential for cysteine-sulfinic acid modifications to modulate the function of various target proteins in a redox-dependent manner. Therefore, at least in some proteins, cysteine-sulfinic acid should be regarded as a post-translational modification rather than simply a type of protein damage.As expected, many of the proteins that are modified by cysteine oxidation are involved in the oxidative stress response or in the maintenance of cellular redox homeostasis. Of these proteins, DJ-1 has special importance in understanding the role of regulatory cysteine oxidation in neuronal survival. Loss of function mutations in DJ-1 are a rare cause of early onset recessive parkinsonism (4, 5), although the exact function of DJ-1 i...

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.

Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson’s disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein.

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.