title: LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.
abstract: Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium. Jean-Marc.Taymans@med.kuleuven.be
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Taymans
authors.forename: Renée
authors.initials: R
authors.lastname: Vancraenenbroeck
authors.forename: Petri
authors.initials: P
authors.lastname: Ollikainen
authors.forename: Alexandra
authors.initials: A
authors.lastname: Beilina
authors.forename: Evy
authors.initials: E
authors.lastname: Lobbestael
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
authors.forename: Veerle
authors.initials: V
authors.lastname: Baekelandt
authors.forename: Mark R
authors.initials: MR
authors.lastname: Cookson
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases