Media, Calcium phosphate transfection kit were obtained from Invitrogen (Carlsbad, CA, USA). Anti-FLAG Affinity Gel Freezer-safe, GTP-agarose, GTP-γ-S, GTP, and ATP were purchased from Sigma (St Louis, MO, USA). Guanosine 5′-triphosphate [γ-32
P], Guanosine 5′-triphosphate [α-32
P] and Adenosine 5′-triphosphate [γ-32
P] were purchased from PerkinElmer (Waltham, MA, USA). Purified Rac I protein was described previously (Guo et al. 2007
). Quickchange Site-Directed Mutagensis kit was purchased from Strategene (Cedar Creek, TX, USA). PEI-F cellulose TLC plates were purchased from J.T. Baker Inc (Phillipsburg, NJ, USA).
Anti-mouse LRRK2 polyclonal antibodies were raised in rabbit against a bacterially expressed glutathione S-transferase(GST)-fusion with LRR domain of mouse LRRK2 (GST-LRR) and were affinity purified by immobilized GST-LRR beads. Anti-FLAG antibody was purchased from Sigma. Anti-tyrosine hydroxylase was obtained from Chemicon (Billerica, MA, USA). Anti-synapsin Ia/b was obtained from BD (San Jose, CA, USA). Anti-synaptophysin, anti-CoxVI and anti-alph-synuclein antibodies were purchased from Abcam (Cambridge, MA, USA). All the FITC and Cy3-conjugated secondary antibodies were obtained from Molecular Probes (Carlsbad, CA, USA).
Generation of FLAG-LRRK2 BAC transgenic mice
BAC clone #RP23-312I9 (240 kb) containing entire mouse LRRK2 genomic sequence (promoter region with at least 35 kb and 3′ non-coding region with 60 kb) was purchased from BAC-PAC (Oakland, CA, USA) resource (CHORI). A nucleotide sequence containing FLAG epitope was inserted in-frame after start codon ‘ATG’ of LRRK2 by BAC modification previously developed in Heintz laboratory (Heintz 2001
). The injection of purified BAC DNA and generation of transgenic mice was done in core facility of Mount Sinai School of Medicine. The BAC transgenic mice expressing FLAG-LRRK2 was genotyped by PCR with primers 5′GACTACAAAGACGATGACGACAAG3′ and 5′CAT-CCACCACCCAGATAATGTC3′.
LRRK2 wild type and mutant cDNA synthesis and HEK-293T cell transfection
Wild type mouse LRRK2 cDNA was synthesized by RT-PCR amplification of several partial sequences covering total LRRK2 proteins from mouse brain total RNA, followed by joining the DNA pieces through DNA restriction enzyme digestion and ligation with DNA ligase. GTPase domain (aa1321–aa1516) was obtained by RT-PCR from mouse brain total RNA. The cDNAs of LRRK2 were confirmed by repetitive sequence. Full length and GTPase domain of LRRK2 are subcloned into p3XFLAG-CMV-7.1 expression vector. The FLAG-LRRK2-full length or GTPase domains were produced in HEK 293T cells after transfection with the above expression vector and purified by using anti-FLAG affinity gel system according to the manual (Sigma). The singe site mutations (R1441C, R1441G and T1398N) within the GTPase coding sequence in p3XFLAG-CMV-ROC were generated by the Quick-change site-directed mutagenesis kit. HEK-293T transfection with above expressing plasmids was performed using standard calcium precipitation method (Invitrogen).
Immuno-purification of FLAG-LRRK2
FLAG-LRRK2-tranfected HEK-293T cells were lysed in lysis buffer (50 mmol/L Tris-HCl at pH7.5, 150 mmol/L NaCl, 1% Triton X100, 1 mmol/L EDTA, 1 mmol/L phenylmethanesulphonylfluoride, 10 μg/mL pepstain and mini complete protease inhibitor cocktail) for 30 min on ice. FLAG-LRRK2 transgenic mouse brain, lung or kidney or WT brain was homogenized in homogenization buffer (20 mmol/L HEPES at pH 7.4, 0.32 mol/L Sucrose, 1 mmol/L NaHCO3, 0.25 mmol/L CaCl2, 1 mmol/L MgCl2, 1 mmol/L PMSF, 10 μg/mL pepstain, and complete protease inhibitor cocktail), then 10% Triton X-100 was added to a final concentration 1% and incubated at 4°C on rotator for 30 min. The lysed cells and homogenized brain were clarified at 12 000 g for 10 min at 4°C, the FLAG-LRRK2 and FLAG-GTPase protein were purified using Anti-FLAG Affinity Gel according the manual (Sigma) with additional extensive wash before elusion. The protein was eluted by using 150 ng/μL FLAG-peptide and stored at −80°C until use.
Kinase assay was carried out for 30 min at 25°C in 30 μL reaction mixture containing 20 mmol/L Tris-HCl at pH 7.5, 15 mmol/L MgCl2, 5 mmol/L EGTA, 20 mmol/L β-glycerol phosphate and 0.1 mg/mL bovine serum albumin (BSA) with [γ-32P]-ATP (6000 cpm/pmol), cold ATP was add to prevent the nonspecific binding. In some cases, assay was done with 3 μg of purified MBP (Myelin basic protein), or α-synuclein as substrate. The reaction was stopped by adding 6XSDS sample buffer, and subjected to SDS-PAGE, stained with Coomassie blue. 32P incorporation was measured by phosphor-imager system (GE Healthcare, Piscataway, NJ, USA).
GTP hydrolysis assay
GTP hydrolysis assay by TLC were performed according to previous report (Xia et al. 2003
) with modification. Briefly, 100 nmol/L purified FLAG-LRRK2 protein and purified Rac1 protein were incubated at 30°C with 20 nmol/L [α-32
P] GTP (3000 cpm/pmol) in reaction buffer (50 mmol/L Tris-HCl pH8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 0.1 mg/mL BSA) for 5 min to load the nucleotides, then Mg2+
was added to a final concentration of 5 mmol/L to stop the loading and start single turnover hydrolysis. The reaction was conducted at 30°C and 5 μL samples was removed at 0, 15, 30, 60, and 120 min and mixed with cooled 5 μL 0.5 mol/L EDTA to stop the hydrolysis reaction. 2 μL mixed samples were spotted and separated by PEI-cellulose TLC plate using 0.5 mol/L KH2
, pH3.4 buffer and quantified with a Storm860 phospho-image analyzer (GE Health-care). Hydrolysis rate (Kcat
) was calculated by fitting the time course of GTP remaining to a single exponential by using Microcal Origin 6.0 (Northampton, MA, USA).
The steady-state GTPase activity was determined by the amounts of [32
P]Pi released using the charcoal assay as previously described (Hart et al. 1990
), 40 nmol/L of purified proteins were incubated for 5, 10, 20, 40, and 60 min at 25°C in a 30 μL of the reaction mixture containing 50 mmol/L Tris-HCl at pH = 8.0, 100 mmol/L NaCl, 1 mmol/L DTT, 1 mmol/L EDTA, 5 mmol/L MgCl2
, 0.1% lubrol, 0.1 mg/mL BSA and 5 μmol/L [γ-32
P]GTP (2000–3000 cpm/pmol). After the incubation, 0.3 mL of ice-cold 5% (w/v) charcoal in 20 mmol/L H3
were added to stop the reaction. The mixture were centrifuged at 10 000 rpm for 10 min at 25°C, the amount of free 32
P release from [γ-32
P] GTP was then estimated by counting the radioactivity of 0.1 mL of the clear supernatant. In some cases, 200 μmol/L ATP was added to the GTP hydrolysis reaction system to prevent the nonspecific binding of GTP to the kinase domain, and the free 32
P released from [γ-32
P] GTP was counted.
For GTP-agarose pull-down, transfected 293Tcells were lysed in lysis buffer (20 mmol/L HEPES at pH 7.4, 2 mmol/L EGTA, 20 mmol/L Glycerol phosphate, 1% Triton X100, 10% glycerol, 1 mmol/L DTT, 1 mmol/L PMSF, 10 μg/mL pepstain, 10 μg/mL aprotinin, 1 mmol/L Na3VO4, and 5 mmol/L NaF) for 30 min on ice, and centrifuged at 12 000 g for 10 min at 4°C. GTP-agarose beads were pre-treated with 1X TBS containing 100 μg/mL BSA for 1 h at 4°C on rotator. 100 μg of clarified cell lysate were incubated with the pre-treated GTP-agarose for 1 h at 4°C on rotator, then added 100 mmol/L GTP and ATP to a final concentration of 2 mmol/L, followed by continuous incubation for additional 2 h. After 2X washing using 500 μL cell lysis buffer, the binding protein was eluted by 20 μL 1X SDS sample buffer and subjected to western blot analysis. The GTPγS binding was determined by filter-binding assay. Briefly, affinity-purified FLAG-tagged proteins were incubated with 5 μmol/L [35S] GTPγS (3000 ~ 8000 cpm/pmol) in binding buffer (20 mmol/L Tris-HCl pH = 8.0, 100 mmol/L NaCl, 5 mmol/L MgCl2, 0.5 mg/mL BSA), at 25°C for 30 min. Reactions were terminated by adding 1 mL ice-cold wash buffer (20 mmol/L Tris-HCl pH = 8.0, 100 mmol/L NaCl, 10 mmol/L MgCl2). The mixtures were filtered through nitrocellulose filters. After washing four times with 1 mL ice-cold wash buffer, the filters were counted by a scintillation counter.
Wild-type and FLAG-LRRK2 transgenic adult mice were perfused with 4% paraformaldehyde in phosphate buffered saline. Brains were dissected, post-fixed overnight in 4% paraformaldehyde and cryoprotected in 30% sucrose and coronal sections of 25 μm were cut on a sliding microtome. The slices were incubated overnight at 4°C with anti-FLAG-(M2) (1: 200, Sigma), affinity purified primary antibody (anti-LRRK2, 1: 1000) or anti-TH antibody (1: 500), followed by incubation with secondary antibodies conjugated to Alexa flourescence dyes (1: 500). The mounted sections were visualized on a Zeiss confocal microscope using fluorescent optics (Carl Zeiss Micro Imaging, Jena, Germany).