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.

Heterogeneity of Leucine-Rich Repeat Kinase 2 Mutations: Genetics, Mechanisms and Therapeutic Implications

“…LRRK2 is an active kinase [12][13][14][15], and, as a member of the ROCO family of proteins [16,17], can both bind and hydrolyse GTP [18][19][20]. It was initially proposed that GTP binding could stimulate the kinase activity of LRRK2, although subsequent results suggested that kinase activity of LRRK2 depends on the ability to bind guanosine nucleotides rather than the GTPbound state [21][22][23], an important mechanistic distinction. Reciprocal regulation of GTP binding by the kinase domain may also occur, as LRRK2 autophosphorylates several residues within the ROC domain.…”

Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors

“…Similarly, mutations in the GTPase domain have been demonstrated to increase kinase activity [57,61], whereas in other studies, they have had little or no effect [34,58]. More recent data have shown that the R1441C/G and Y1699C mutations reduced the GTPase activity that in turn regulates kinase activity [14,15]. The majority of studies have used either recombinantly expressed and purified LRRK2 [58] or immunoprecipitated LRRK2 from recombinant mammalian expression systems [34,57,61] and investigated autophosphorylation or the use of surrogate substrates (myelin basic protein, LRRKtide, NICtide), all which are relatively poor substrates under those assay conditions.…”

“…A number of novel variants have been identified in this gene in PD patients, but only seven of these (N1437H, R1441C, R1441G, S1761R, Y1699C, G2019S, and I2020T) can be considered as definitively disease causing, on the basis of co-segregation with disease in families, and an absence in controls [5,[11][12][13]. These mutations either lead to an increased kinase activity (G2019S and I2020T) or to a reduced GTPase activity (R1441C/G and Y1699C), which in turn regulates kinase activity [14][15][16] (vide infra). The G2019S mutation is relatively frequent in some populations from Southern Europe and in certain populations, such as the Ashkenazi Jews and North African Berber Arabs, where the prevalence can be as high as 40% [8].…”

Modeling LRRK2 Pathobiology in Parkinson’s Disease: From Yeast to Rodents

“…LRRK2 autophosphorylation may serve a regulatory function and occurs at residues within or adjacent to the ROC GTPase domain Webber et al 2011;Kamikawaji et al 2013). The GTPase domain of LRRK2 binds guanine nucleotides and is capable of hydrolyzing GTP at a slow rate, apparently independent of its oligomerization state (Ito et al 2007; Lewis et al 2007;Taymans et al 2011;Biosa et al 2013;Liao et al 2014). Although there is evidence for a functional interplay between the two enzymatic domains, the biochemical mechanisms governing LRRK2 enzymatic functions remain unclear.…”

“…Although there is evidence for a functional interplay between the two enzymatic domains, the biochemical mechanisms governing LRRK2 enzymatic functions remain unclear. Interestingly, GTP hydrolysis and GTP binding activities of LRRK2 are both required for LRRK2 kinase activity, whereas the contribution of LRRK2 autophosphorylation within the GTPase domain to GTP binding and GTP hydrolysis activities is incompletely understood (Ito et al 2007;West et al 2007;Taymans et al 2011;Biosa et al 2013). Kinase-inactive variants of LRRK2 exhibit normal GTP binding and GTP hydrolysis activities, although mutation of individual autophosphorylation sites within the GTPase domain (i.e., T1503) can alter kinase activity (Webber et al 2011;Biosa et al 2013).…”