Mutations in a novel gene encoding the leucine-rich repeat kinase 2 (LRRK2) have recently been shown to be the most common cause of autosomal dominant, late-onset Parkinson's disease. However, little is known about this multi-domain complex protein, LRRK2, that has several functionally important domains, including a Ras-related GTPase domain and a MAPKKK (mitogen- activated protein kinase kinase kinase) domain. Genetic studies have identified two pathogenic mutations, R1441C within the GTPase domain, and G2019S within the MAPKKK domain. Therefore, pathogenic LRRK2 mutations specifically target the GTPase "and MAPKKK activities of LRRK2 in causing familial PD. As both the GTPase and MAPKKK are located upstream of MAP kinase signal transduction cascades, LRRK2 is likely to serve as a dual regulatory switch controlling critical cellular processes relevant to the pathogenesis of Parkinson's disease. Our long-term objective is to elucidate the pathogenic mechanisms mediated by the disease-causing LRRK2 mutations. In this application, we propose to investigate the structure-activity relationship between the R1441C and G2019S mutations and the catalytic activities of LRRK2. Two specific aims are designed to focus on the analyses of the effects of the pathogenic mutations on GTPase and MAPKKK activities of LRRK2, and their associated substrates and binding-partners in human neuronal cell lines. Transfected cell lines expressing FLAG epitope-tagged LRRK2 carrying the R1441C and G2019S mutations within the respective GTPase and MAPKKK domains will be established. In Specific Aim 1, we will examine the effect of R1441C mutation on LRRK2 GTPase activity in terms of guanine nucleotide binding and hydrolysis, co-factor interactions, and potential activation of nearby MAPKKK domain. In Specific Aim 2, we will investigate the effect of G2019S mutation on LRRK2 kinase catalysis, phosphorylation of cellular substrates, and activation of known MAP kinase pathways. These proposed experiments will likely yield essential data that provide a structural basis for the pathogenic LRRK2 mutations in initiating the disease process. The proof-of- principle research proposed here is also likely to lead to a much-needed breakthrough in our understanding of the pathogenic roles of LRRK2 in the cellular mechanisms underlying the pathogenesis of Parkinson's disease. [unreadable] [unreadable] [unreadable]