Leucine‐rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson’s disease R1441C/G mutants

Abstract

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson's disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD. Keywordsbacterial artificial chromosome transgenics; GTPase; kinase; Leucine-rich repeat kinase 2; Parkinson's disease Parkinson's disease (PD) is the second most common neurodegenerative disease. It is characterized clinically by a movement disorder that includes rigidity, resting tremor and bradykinesia, and pathologically by degeneration of dopamine neurons in the substantia nigra pars compacta as well as other selected brain regions. The etiology in most cases is unknown, but during the past several years, a number of gene mutations have been identified in some familial and sporadic forms of PD that provide an opportunity to elucidate the molecular mechanisms underlying the pathogenesis of cell death in PD. Recent studies suggest that

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.…”

Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson's disease?

“…The GTPase activity for N1437H has not been determined, but it has been shown that this mutant has increased GTP binding [13], which is consistent with lower GTPase activity. The reported GTP-binding capacity of R1441C/G and Y1699C is less consistent, with some reports of an increase and others finding no change in GTP binding [45,47,48]. These effects are probably related, as it has been proposed that the ROC and COR domains may interact with each other.…”

Update on the Functional Biology of Lrrk2

“…In mice, during embryonic development LRRK2 mRNA expression is not detectable before E15 after which it steadily rises, suggesting it is not an essential developmental gene [31]. Lrrk2 protein is evident by E17 [31] and continues to rise progressively and peaks around P60 [33]. Levels of Lrrk2 remain constant in rodents during aging [34].…”

“…Outside of the CNS, high expression is found in the lungs, kidney, spleen and leucocytes [3,[31][32][33][34]. In mice, during embryonic development LRRK2 mRNA expression is not detectable before E15 after which it steadily rises, suggesting it is not an essential developmental gene [31].…”

“…Several groups now have shown that Lrrk2 can bind GTP via GTP-sepharose or GTPγS-binding assays, whereas disruption of the P-loop in the nucleotide-binding pocket of the Roc domain abolishes GTP binding [33,47,53,[57][58][59][60]. GTPase activity has been more difficult to demonstrate [57,60], presumably owing to the requirement of GEFs and GAPs for in vitro assays; however, optimized experimental conditions have now enabled GTPase activity, albeit low, to be detected [33,53,58,59].…”