The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity

Abstract

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal dominant late-onset Parkinson disease (PD). The LRRK2 protein consists of multiple domains and belongs to the Roco family, a novel group of the Ras/GTPase superfamily. Besides the GTPase (Roc) domain, it contains a predicted kinase domain, with homology to MAP kinase kinase kinases. Using cell fractionation and immunofluorescence microscopy, we show that LRRK2 is localized in the cytoplasm and is associated with cellular membrane structures. The purified LRRK2 protein demonstrates autokinase activity. The disease-associated I2020T mutant shows a significant increase in autophosphorylation of approximately 40% in comparison to wild-type protein in vitro. This suggests that the pathology of PD caused by the I2020T mutation is associated with an increase rather than a loss in LRRK2 kinase activity.

Update on the Functional Biology of Lrrk2

“…In addition, Lrrk2 is found in lipid rafts, microdomains on the cellular membrane of membranous organelles for example, golgi apparatus, plasma membrane, synaptic vesicles, mitochondria and endoplasmic reticulum [48]. However, it is thought to be membrane associated rather than membrane integrated [44,46,48]. Similar results were observed in fractionation of rat primary cultures, where Lrrk2 punctate structures particularly colocalize to mitochondria and lysosomal markers [40].…”

“…When transfected in a variety of cell lines, Lrrk2 is largely excluded from the nucleus and is present in diffuse localization throughout the cytoplasm [20,[44][45][46][47], possibly in association with mitochondria [44,46]. Although Lrrk2 does not contain any hydrophobic transmembrane domains or obvious mitochondrial or nuclear-targeting sequences [46], subcellular fractionation reveals Lrrk2 in membranous and microsomal fractions [44,46,48].…”

“…Although Lrrk2 does not contain any hydrophobic transmembrane domains or obvious mitochondrial or nuclear-targeting sequences [46], subcellular fractionation reveals Lrrk2 in membranous and microsomal fractions [44,46,48]. In addition, Lrrk2 is found in lipid rafts, microdomains on the cellular membrane of membranous organelles for example, golgi apparatus, plasma membrane, synaptic vesicles, mitochondria and endoplasmic reticulum [48].…”

Ancient Origin of the Parkinson Disease Gene LRRK2

“…In humans, however, it is unknown whether loss-of-function mutations in LRRK2 lead to dopaminergic cell death. On the contrary, it seems that it is increased/constitutive activity of the LRRK2 protein (West et al 2005;Gloeckner et al 2006;Smith et al 2006;Guo et al 2007;Li et al 2007). In summary, researchers in this field should be very careful before extrapolating results obtained for protostome LRRK genes to humans.…”

Progress in the pathogenesis and genetics of Parkinson's disease

“…The ROC domain is able to bind GTP but it does not have GTPase activity, but GTP binding is essential for the MAPKKK domain to exert kinase activity (Ito et al 2007); some of the mutant LRRK2 have increased kinase activity (Gloeckner et al 2006). Other functional domains are believed to be important in protein-protein interactions (Zimprich et al 2004).…”