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.
Highlights
Leucine rich repeat kinase 2 (LRRK2) has been identified as a Parkinson’s disease (PD) gene responsible for parkinsonism with a clinical course essentially identical to that in idiopathic PD [1,2]
LRRK2 kinase activity of the affinity purified protein is not altered upon co-incubation or preloading with guanine nucleotides
Via metabolic labeling and thin layer chromatography analysis, we found that our stringent purification procedure yielded protein devoid of guanine nucleotides
Summary
Leucine rich repeat kinase 2 (LRRK2) has been identified as a Parkinson’s disease (PD) gene responsible for parkinsonism with a clinical course essentially identical to that in idiopathic PD [1,2]. Potential protein-protein interaction regions include an ankyrin repeat domain, a leucine rich repeat domain and a WD40 domain as well as two catalytic domains including a GTPase domain of the Ras of complex proteins family (ROC) and a kinase domain of the tyrosine kinase like family [3,4]. Ras family GTPases and tyrosine kinase like kinases are often associated as elements of the same intracellular signaling pathway, suggesting a functional interaction between both of these catalytic functions within LRRK2. Ras GTPases act as molecular switches cycling between a guanosine triphosphate (GTP) bound ‘on’ state and a guanosine diphosphate (GDP) bound ‘off’ state. In the ‘on’ state, Ras GTPases activate an effector protein such as a kinase via direct binding
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