Characterization of TAE684 as a potent LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.     

(G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD

(G2019S) mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurons and parkinsonism phenotypes of motor dysfunction. LRRK2 is a member of mixed lineage kinase subfamily of mitogen-activated protein kinase kinase kinases (MAPKKKs). We hypothesized that (G2019S) mutation augmented LRRK2 kinase activity, leading to overphosphorylation of downstream MAPK kinase (MKK) and resulting in activation of neuronal death signal pathway. Consistent with our hypothesis, (G2019S) LRRK2 expressed in HEK 293 cells exhibited an augmented kinase activity of phosphorylating MAPK kinase 4 (MKK4) at Ser(257), and protein expression of active phospho-MKK4(Ser257) was upregulated in the SN of (G2019S) LRRK2 transgenic mice. Protein level of active phospho-JNK(Thr183/Tyr185) and phospho-c-Jun(Ser63), downstream targets of phospho-MKK4(Ser257), was increased in the SN of (G2019S) LRRK2 mice. Upregulated mRNA expression of pro-apoptotic Bim and FasL, target genes of phospho-c-Jun(Ser63), and formation of active caspase-9, caspase-8 and caspase-3 were also observed in the SN of (G2019S) LRRK2 transgenic mice. Our results suggest that mutant (G2019S) LRRK2 activates MKK4-JNK-c-Jun pathway in the SN and causes the resulting degeneration of SNpc dopaminergic neurons in PD transgenic mice.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species     

Triazolopyridazine LRRK2 kinase inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson’s disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of [1,2,4]triazolo[4,3-b]pyridazines that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays and show an unprecedented selectivity towards the G2019S mutant. A structural rational for the observed selectivity is proposed.     

High LRRK2 levels fail to induce or exacerbate neuronal alpha-synucleinopathy in mouse brain

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson's disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy.     

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.     

Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation

Background

Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.

Methodology/Principal Findings

Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.

Conclusions/Significance

We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and {"type":"entrez-nucleotide","attrs":{"text":"GW441756","term_id":"315858226","term_text":"GW441756"}}GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.     

Efficient allele-specific targeting of LRRK2 R1441 mutations mediated by RNAi

Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.     

Discovery of 4-alkylamino-7-aryl-3-cyanoquinoline LRRK2 kinase inhibitors

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson’s disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.     

Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond

The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson’s disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases. Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2 kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective inhibitor of the present series, is characterized by an IC₅₀ of 15 nM against wild-type LRRK2 and 10 nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled toxicity determination in whole organisms.     

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.     

IT leucine-rich repeat kinase 2, the causative mutant molecule of familial Parkinson’s disease, has a higher intracellular degradation rate than the wild-type molecule

Leucine-rich repeat kinase 2 (LRRK2) has been identified as the causal gene for autosomal dominant familial Parkinson’s disease (PD), although the mechanism of neurodegeneration involving the mutant LRRK2 molecules remains unknown. In the present study, we found that the protein level of transfected I²⁰²⁰T mutant LRRK2 was significantly lower than that of wild-type and G²⁰¹⁹S mutant LRRK2, although the intracellular localization of the I²⁰²⁰T and wild-type molecules did not differ. Pulse-chase experiments proved that the I²⁰²⁰T LRRK2 molecule has a higher degradation rate than wild-type or G²⁰¹⁹S LRRK2. Upon addition of proteasome and lysosome inhibitors, the protein level of I²⁰²⁰T mutant LRRK2 reached that of the wild-type. These results indicate that I²⁰²⁰T mutant LRRK2 is more susceptible to post-translational degradation than the wild-type molecule. Our results indicate a novel molecular feature characteristic to I²⁰²⁰T LRRK2, and provide a new insight into the mechanism of neurodegeneration caused by LRRK2.     

Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO^LRRK2 self-interaction. Interestingly, ROCO^LRRK2 fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO^LRRK2 reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO^LRRK2 fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.     

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson’s disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.     

Mutant LRRK2 enhances glutamatergic synapse activity and evokes excitotoxic dendrite degeneration

Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinson's disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.     

Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.     

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.     

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.     

LRRK2 directly phosphorylates Akt1 as a possible physiological substrate: impairment of the kinase activity by Parkinson's disease-associated mutations

LRRK2 is the causal molecule for autosomal-dominant familial Parkinson's disease, although its true function, including its physiological substrates, remains unknown. Here, using in vitro kinase assay with recombinant proteins, we demonstrated for the first time that LRRK2 directly phosphorylates Akt1, a central molecule involved in signal transduction for cell survival and prevention of apoptosis. Ser473, one of two amino acids essential for Akt1 activation, was the target site for LRRK2. A knockdown experiment using intact cells also demonstrated LRRK2-mediated phosphorylation of Akt1 (Ser473), suggesting that Akt1 is a convincing candidate for the physiological substrate of LRRK2. The disease-associated mutations, R1441C, G2019S, and I2020T, exhibited reduced interaction with, and phosphorylation of, Akt1, suggesting one possible mechanism for the neurodegeneration caused by LRRK2 mutations.