Name: Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag (Video) | JoVE Journal
Uploaded: 2017-12-14T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Learn to detect Rab10 phosphorylation by LRRK2 using SDS-PAGE with a phosphate-binding tag. This method enables estimation of phosphorylation stoichiometry in Parkinson's disease research.

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We thank Dr. Takeshi Iwatsubo (University of Tokyo, Japan) for kindly providing the plasmids encoding 3xFLAG-LRRK2 WT and mutants. We also thank Dr. Dario Alessi (University of Dundee, UK) for kindly providing MLi-2 and the plasmid encoding HA-Rab10. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP17K08265 (G.I.).

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</ol>

The authors have nothing to disclose.

Here, we describe a facile and robust method of detecting Rab10 phosphorylation by LRRK2 at endogenous levels based on the P-tag methodology. Because the currently available antibody against phosphorylated Rab10 works only with overexpressed proteins15, the present method utilizing P-tag SDS-PAGE is the only way to assess endogenous levels of Rab10 phosphorylation. Moreover, the present method allows the estimation of the stoichiometry of Rab10 phosphorylation in cells. Because the P-tag methodolo...

PD is one of the most common neurodegenerative diseases, predominantly affecting dopaminergic neurons in the midbrain, resulting in dysfunction of the motor systems in elderly people1. While most patients develop PD in a sporadic manner, there are families inheriting the disease. Mutations in several genes have been found to be linked with FPD2. One of the causative genes for FPD is LRRK2, in which eight missense mutations (N1437H, R1441C/G/H/S, Y1699C, G2019S, and I2020T) linked to a dominantly inherited FPD called PARK8 have so far been reported3,4,5. Several genome-wide association studies (GWAS) of sporadic PD patients have also identified genomic variations at the LRRK2 locus as a risk factor for PD, suggesting that abnormality in the function of LRRK2 is a common cause of neurodegeneration in both sporadic and PARK8 FPD6,7,8.
LRRK2 is a large protein (2,527 amino acids) consisting of a leucine-rich repeat domain, a GTP-binding Ras of complex proteins (ROC) domain, a C-terminal of ROC (COR) domain, a serine/threonine protein kinase domain, and a WD40 repeat domain9. The eight FPD mutations locate in these functional domains; N1437H and R1441C/G/H/S in the ROC domain, Y1699C in the COR domain, G2019S and I2020T in the kinase domain. Since G2019S mutation, which is the most frequently found mutation in PD patients10,11,12, increases the kinase activity of LRRK2 by 2 - 3 fold in vitro13, it is hypothesized that the abnormal increase in phosphorylation of LRRK2 substrate(s) is toxic to neurons. However, it has been impossible to study whether the phosphorylation of physiologically relevant LRRK2 substrates is altered in familial/sporadic PD patients due to the lack of methods evaluating it in patient derived samples.
Protein phosphorylation is generally detected by immunoblotting or enzyme-linked immunosorbent assay (ELISA) using antibodies specifically recognizing the phosphorylated state of proteins or by mass spectrometric analysis. However, the former strategy sometimes cannot be applied because of the difficulties in creating phosphorylation-specific antibodies. Metabolic labeling of cells with radioactive phosphate is another option to examine physiological levels of phosphorylation when phosphorylation-specific antibodies are not readily available. However, it requires a large amount of radioactive materials and therefore involves some specialized equipment for radioprotection14. Mass spectrometric analysis is more sensitive compared to these immunochemical methods and became popular in analyzing protein phosphorylation. However, the sample preparation is time-consuming, and expensive instruments are required for the analysis.
A subset of the Rab GTPase family including Rab10 and Rab8 was recently reported as direct physiological substrates for LRRK2 based on the result of a large-scale phosphoproteomic analysis15. We then demonstrated that Rab10 phosphorylation was increased by FPD mutations in mouse embryonic fibroblasts and in the lungs of knockin mice16. In this report, we chose to employ a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-based method in which a P-tag molecule is co-polymerized into SDS-PAGE gels (P-tag SDS-PAGE) for detecting the endogenous levels of Rab10 phosphorylation, because a highly sensitive antibody specific for phosphorylated Rab10 was still lacking. We have failed to detect the phosphorylation of endogenous Rab8 due to the poor selectivity of currently available antibodies for total Rab8. Therefore, we decided to focus on the Rab10 phosphorylation. LRRK2 phosphorylates Rab10 at Thr73 locating at the middle of the highly conserved &#34;switch II&#34; region. High conservation of the phosphorylation sites among Rab proteins might be one of the reasons why phosphospecific antibodies recognizing distinct Rab proteins are difficult to make.
The phosphorylation of Rab8A by LRRK2 inhibits the binding of Rabin8, a guanine nucleotide exchange factor (GEF) which activates Rab8A by exchanging the bound GDP with GTP15. Phosphorylation of Rab10 and Rab8A by LRRK2 also inhibits the binding of GDP-dissociation inhibitors (GDIs), which are essential to the activation of Rab proteins by extracting GDP-bound Rab proteins from membranes15. Collectively, it is hypothesized that the phosphorylation of Rab proteins by LRRK2 prevents them from activation although the precise molecular mechanism and physiological consequences of the phosphorylation remain unclear.
P-tag SDS-PAGE was invented by Kinoshita et al. in 2006: In this method, acrylamide was covalently coupled with P-tag, a molecule capturing phosphates with high affinity, which copolymerized into SDS-PAGE gels17. Because the P-tag molecules in a SDS-PAGE gel selectively retard electrophoretic mobility of phosphorylated proteins, P-tag SDS-PAGE can separate phosphorylated proteins from non-phosphorylated ones (Figure 1). If the protein-of-interest is phosphorylated on multiple residues, a ladder of bands corresponding to differentially phosphorylated forms will be observed. In the case of Rab10, we observe only one shifted band, indicating that Rab10 is phosphorylated only at Thr73. The major advantage of P-tag SDS-PAGE over immunoblotting with phosphorylation-specific antibodies is that phosphorylated Rab10 can be detected by immunoblotting with non-phosphorylation-specific antibodies (i.e., recognizing total Rab10) after being transferred on membranes, which is generally more specific, sensitive, and available from commercial/academic sources. Another advantage of using P-tag SDS-PAGE is that one can obtain approximate estimation of the stoichiometry of phosphorylation, which is impossible by immunoblotting with phosphorylation-specific antibodies or by metabolic labeling of cells with radioactive phosphates.
Apart from the use of inexpensive P-tag acrylamide and some minor modifications related to it, the present method for detection of Rab10 phosphorylation by LRRK2 follows a general protocol of immunoblotting. Therefore, it should be straightforward and easily executable in any laboratories where immunoblotting is a usual practice, with any types of samples including purified proteins, cell lysates, and tissue homogenates.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>homemade</td><td></td><td>150 mM NaCl, 8 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-12H&lt;sub&gt;2&lt;/sub&gt;O, 2.7 mM KCl, 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;in MilliQ water and sterilized by autoclaving</td></tr><tr><td>Sodium chloride</td><td>Nacalai Tesque</td><td>31320-34</td><td></td></tr><tr><td>Sodium Disodium Hydrogenphosphate 12-Water</td><td>Wako</td><td>196-02835</td><td></td></tr><tr><td>Potassium chloride</td><td>Wako</td><td>163-03545</td><td></td></tr><tr><td>Potassium Dihydrogen Phosphate</td><td>Wako</td><td>169-04245</td><td></td></tr><tr><td>2.5% Trypsin (10X)</td><td>Sigma-Aldrich</td><td>T4549</td><td>Dilute 10-fold with sterile DPBS for preparing working solution</td></tr><tr><td>Dulbecco&#039;s modified Eagle medium&lt;br/&gt; (DMEM)</td><td>Wako</td><td>044-29765</td><td></td></tr><tr><td>Fetal bovine serum</td><td>BioWest</td><td>S1560</td><td>Heat-inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Wako</td><td>168-23191</td><td></td></tr><tr><td>HEPES</td><td>Wako</td><td>342-01375</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Wako</td><td>198-13765</td><td></td></tr><tr><td>Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000)</td><td>PolySciences, Inc.</td><td>24765-1</td><td>Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH</td></tr><tr><td>Dimethyl sulfoxide</td><td>Wako</td><td>045-28335</td><td></td></tr><tr><td>Tris</td><td>STAR</td><td>RSP-THA500G</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako</td><td>080-01066</td><td></td></tr><tr><td>Polyoxyethylene(10) Octylphenyl Ether</td><td>Wako</td><td>160-24751</td><td>Equivalent to Triton X-100</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetraacetic acid (EGTA)</td><td>Wako</td><td>346-01312</td><td></td></tr><tr><td>Sodium orthovanadate(V)</td><td>Wako</td><td>198-09752</td><td></td></tr><tr><td>Sodium fluoride</td><td>Kanto Chemical</td><td>37174-20</td><td></td></tr><tr><td>&amp;beta;-Glycerophosphoric Acid Disodium Salt Pentahydrate</td><td>Nacalai Tesque</td><td>17103-82</td><td></td></tr><tr><td>Sodium pyrophosphate decahydrate</td><td>Kokusan Chemical</td><td>2113899</td><td></td></tr><tr><td>Microcystin-LR</td><td>Wako</td><td>136-12241</td><td></td></tr><tr><td>Sucrose</td><td>Wako</td><td>196-00015</td><td></td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td>Dissolve one tablet in 1 mL water, which can be stored at -20 &amp;deg;C for a month. Use it at 1:50 dilution for cell lysis</td></tr><tr><td>Pierce Coomassie (Bradford) Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23200</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Nacalai Tesque</td><td>31607-65</td><td></td></tr><tr><td>Glycerol</td><td>Wako</td><td>075-00616</td><td></td></tr><tr><td>Bromophenol blue</td><td>Wako</td><td>021-02911</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Kanto Chemical</td><td>25099-00</td><td></td></tr><tr><td>Ethanol</td><td>Wako</td><td>056-06967</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>136-01837</td><td></td></tr><tr><td>Phosphate-binding tag acrylamide</td><td>Wako</td><td>AAL-107</td><td>P-tag acrylamide</td></tr><tr><td>40% (w/v) acrylamide solution</td><td>Nacalai Tesque</td><td>06119-45</td><td>Acrylamide:Bis = 29:1</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Nacalai Tesque</td><td>33401-72</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Wako</td><td>016-08021</td><td>10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water</td></tr><tr><td>2-propanol</td><td>Wako</td><td>166-04831</td><td></td></tr><tr><td>Manganese chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M3634</td><td></td></tr><tr><td>Precision Plus Protein Prestained Standard</td><td>Bio-Rad</td><td>1610374, 1610373, 1610377</td><td>Molecular weight marker used in the protocol</td></tr><tr><td>WIDE-VIEW Prestained Protein Size Marker III</td><td>Wako</td><td>230-02461</td><td></td></tr><tr><td>Glycine</td><td>Nacalai Tesque</td><td>17109-64</td><td></td></tr><tr><td>Amersham Protran NC 0.45</td><td>GE Healthcare</td><td>10600007</td><td>Nitrocellulose membrane</td></tr><tr><td>Durapore Membrane Filter</td><td>EMD Millipore</td><td>GVHP00010</td><td>PVDF membrane</td></tr><tr><td>Filter Papers No.1</td><td>Advantec</td><td>00013600</td><td></td></tr><tr><td>Ponceau S</td><td>Nacalai Tesque</td><td>28322-72</td><td></td></tr><tr><td>Acetic acid</td><td>Wako</td><td>017-00251</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td>polyoxyethylenesorbitan monolaurate</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Wako</td><td>345-01865</td><td></td></tr><tr><td>Skim milk powder</td><td>Difco Laboratories</td><td>232100</td><td></td></tr><tr><td>Immunostar</td><td>Wako</td><td>291-55203</td><td>ECL solution (Normal sensitivity)</td></tr><tr><td>Immunostar LD</td><td>Wako</td><td>290-69904</td><td>ECL solution (High sensitivity)</td></tr><tr><td>CBB staining solution</td><td>homemade</td><td></td><td>1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water</td></tr><tr><td>CBB R-250</td><td>Wako</td><td>031-17922</td><td></td></tr><tr><td>CBB destaining solution</td><td>homemade</td><td></td><td>12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-HA antibody</td><td>Sigma-Aldrich</td><td>11583816001</td><td>Used at 0.2 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-Rab10 antibody</td><td>Cell Signaling Technology</td><td>#8127</td><td>Used at 1:1000 for immunoblotting.&lt;br/&gt; Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016.</td></tr><tr><td>anti-pSer935 antibody</td><td>Abcam</td><td>ab133450</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-LRRK2 antibody</td><td>Abcam</td><td>ab133518</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody</td><td>Sigma-Aldrich</td><td>T9026</td><td>Used at 1 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>anti-GAPDH antibody</td><td>Santa-Cruz</td><td>sc-32233</td><td>Used at 0.02 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>515-035-003</td><td>Used at 0.16 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-035-003</td><td>Used at 0.16 &amp;mu;g/mL for immunoblotting.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Inhibitors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GSK2578215A</td><td>MedChem Express</td><td>HY-13237</td><td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &amp;deg;C</td></tr><tr><td>MLi-2</td><td>Provided by Dr Dario Alessi (University of Dundee)</td><td></td><td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rab10/pcDNA5 FRT TO HA</td><td>Provided by Dr Dario Alessi&lt;br/&gt; (University of Dundee)</td><td></td><td>This plasmid expresses amino-terminally HA-tagged human Rab10.</td></tr><tr><td>LRRK2 WT/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Ito et al., Biochemistry, 46: 1380&amp;ndash;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2.</td></tr><tr><td>LRRK2 K1906M/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Ito et al., Biochemistry, 46: 1380&amp;ndash;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2.</td></tr><tr><td>LRRK2 N1437H/p3xFLAG-CMV-10</td><td></td><td></td><td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441C/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441G/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441H/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 R1441S/p3xFLAG-CMV-10</td><td></td><td></td><td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 Y1699C/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 G2019S/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2.</td></tr><tr><td>LRRK2 I2020T/p3xFLAG-CMV-10</td><td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td><td></td><td>Kamikawaji et al., Biochemistry, 48: 10963&amp;ndash;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific</td><td>Forma Series II 3110 Water-Jacketed</td><td></td></tr><tr><td>Auto Pipette</td><td>Drummond</td><td>Pipet-Aid PA-400</td><td></td></tr><tr><td>Micropipette P10</td><td>Nichiryo</td><td>00-NPX2-10</td><td>0.5&amp;ndash;10 &amp;mu;L</td></tr><tr><td>Micropipette P200</td><td>Nichiryo</td><td>00-NPX2-200</td><td>20&amp;ndash;200 &amp;mu;L</td></tr><tr><td>Micropipette P1000</td><td>Nichiryo</td><td>00-NPX2-1000</td><td>100&amp;ndash;1000 &amp;mu;L</td></tr><tr><td>Tips for micropipette P10</td><td>STAR</td><td>RST-481LCRST</td><td>Sterile</td></tr><tr><td>Tips for micropipette P200</td><td>FUKAEKASEI</td><td>1201-705YS</td><td>Sterile</td></tr><tr><td>Tips for micropipette P1000</td><td>STAR</td><td>RST-4810BRST</td><td>Sterile</td></tr><tr><td>5 mL disporsable pipette</td><td>Greiner</td><td>606180</td><td>Sterile</td></tr><tr><td>10 mL disporsable pipette</td><td>Greiner</td><td>607180</td><td>Sterile</td></tr><tr><td>25 mL disporsable pipette</td><td>Falcon</td><td>357535</td><td>Sterile</td></tr><tr><td>Hematocytometer</td><td>Sunlead Glass</td><td>A126</td><td>Improved Neubeuer</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CKX53</td><td></td></tr><tr><td>10 cm dishes</td><td>Falcon</td><td>353003</td><td>For tissue culture</td></tr><tr><td>6-well plates</td><td>AGC Techno Glass</td><td>3810-006</td><td>For tissue culture</td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>Vortex-Genie 2</td><td></td></tr><tr><td>Cell scrapers</td><td>Sumitomo Bakelite</td><td>MS-93100</td><td></td></tr><tr><td>1.5 mL tubes</td><td>STAR</td><td>RSV-MTT1.5</td><td></td></tr><tr><td>15 mL tubes</td><td>AGC Techno Glass</td><td>2323-015</td><td></td></tr><tr><td>50 mL tubes</td><td>AGC Techno Glass</td><td>2343-050</td><td></td></tr><tr><td>Centrifuges</td><td>TOMY</td><td>MX-307</td><td></td></tr><tr><td>96-well plates</td><td>Greiner</td><td>655061</td><td>Not for tissue culture</td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>SpectraMax M2e</td><td></td></tr><tr><td>SDS&amp;ndash;PAGE tanks</td><td>Nihon Eido</td><td>NA-1010</td><td></td></tr><tr><td>Transfer tanks</td><td>Nihon Eido</td><td>NA-1510B</td><td></td></tr><tr><td>Gel plates (notched)</td><td>Nihon Eido</td><td>NA-1000-1</td><td></td></tr><tr><td>Gel plates (plain)</td><td>Nihon Eido</td><td>NA-1000-2</td><td></td></tr><tr><td>Silicon spacers</td><td>Nihon Eido</td><td>NA-1000-16</td><td></td></tr><tr><td>17-well combs</td><td>Nihon Eido</td><td>Custom made</td><td></td></tr><tr><td>Binder clips</td><td>Nihon Eido</td><td>NA-1000-15</td><td></td></tr><tr><td>5 mL syringe</td><td>Terumo</td><td>SS-05SZ</td><td></td></tr><tr><td>21G</td><td>Terumo</td><td>NN-2138R</td><td></td></tr><tr><td>Power Station 1000 VC</td><td>ATTO</td><td>AE-8450</td><td>Power supply for SDS&amp;ndash;PAGE and transfer</td></tr><tr><td>Large weighing boats</td><td>Ina Optika</td><td>AS-DL</td><td></td></tr><tr><td>Plastic containers</td><td>AS ONE</td><td>PS CASE No.4</td><td>10 x 80 x 50 mm</td></tr><tr><td>Rocking shaker</td><td>Titech</td><td>NR-10</td><td></td></tr><tr><td>Styrene foam box</td><td>generic</td><td></td><td>The internal dimensions should fit one transfer tank (200 x 250 x 250 mm).</td></tr><tr><td>ImageQuant LAS-4000</td><td>GE Healthcare</td><td></td><td>An imager equipped with a cooled CCD camera for detection of ECL</td></tr></tbody></table>

rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

Overexpression System: Phosphorylation of HA-Rab10 by 3×FLAG-LRRK2 in HEK293 Cells:
HEK293 cells were transfected with 0.266 µg of HA-Rab10 wild-type and 1.066 µg of 3×FLAG-LRRK2 (wild-type, kinase-inactive mutant (K1906M), or FPD mutants). Rab10 phosphorylation was examined by P-tag SDS-PAGE followed by immunoblotting using an anti-HA antibody (Figure 2). 10 µg of proteins were run on a 10% gel (80 x ...

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

rab10-phosphorylation-detection-lrrk2-activity-using-sds-page-with

Research

JoVE Journal

Biochemistry

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction...

Assaying the Kinase Activity of LRRK2 in vitro

This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Oligopeptide Competition Assay for Phosphorylation Site Determination

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by γ[32P] ATP labeling.

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

This protocol describes the rapid and highly sensitive quantification of Förster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Learn to detect Rab10 phosphorylation by LRRK2 using SDS-PAGE with a phosphate-binding tag. This method enables estimation of phosphorylation stoichiometry in Parkinson's disease research.

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

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