title: GTPase activity regulates kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2.
abstract: 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.
authors.affiliation: Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Fe´de´ rale de Lausanne (EPFL), Lausanne 1015, Switzerland.
authors.forename: Alice
authors.initials: A
authors.lastname: Biosa
authors.forename: Alzbeta
authors.initials: A
authors.lastname: Trancikova
authors.forename: Laura
authors.initials: L
authors.lastname: Civiero
authors.forename: Liliane
authors.initials: L
authors.lastname: Glauser
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
authors.forename: Elisa
authors.initials: E
authors.lastname: Greggio
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Moore
mesh_terms: D000818:Animals; D019281:Dimerization; D005260:Female; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D020125:Mutation, Missense; D009474:Neurons / Q000201:enzymology; D010300:Parkinson Disease / Q000201:enzymology* / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D006150:Guanine Nucleotides; D018841:HSP90 Heat-Shock Proteins; D006160:Guanosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases