Name: rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag
Uploaded: 2017-12-14T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

岩坪威博士 (東京大学、日本) にありがとう親切プラスミド 3xFLAG LRRK2 WT と変異体を提供します。我々 も博士ダリオ ・ アレッシ (、英国ダンディー大学) 提供いただきありがとうございます親切 MLi 2 とエンコード HA Rab10 プラスミド。この作品は、科学の振興 (JSPS) 科研費助成番号 JP17K08265 (GI) の日本社会によって支えられました。

<ol>
	<li>Sveinbjornsdottir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+symptoms+of+Parkinson's+disease.">The clinical symptoms of Parkinson's disease.</a> J. Neurochem. 139 (Suppl. 1), 318-324 (2016).</li><li>Hernandez, D. G., Reed, X., Singleton, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+in+Parkinson+disease:+Mendelian+versus+non-Mendelian+inheritance.">Genetics in Parkinson disease: Mendelian versus non-Mendelian inheritance.</a> J. Neurochem. 139 (Suppl. 1), 59-74 (2016).</li><li>Pais&#225;n-Ru&#237;z, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+gene+containing+mutations+that+cause+PARK8-linked+Parkinson's+disease.">Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.</a> Neuron. 44 (4), 595-600 (2004).</li><li>Zimprich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+LRRK2+cause+autosomal-dominant+parkinsonism+with+pleomorphic+pathology.">Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.</a> Neuron. 44 (4), 601-607 (2004).</li><li>Gilks, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+LRRK2+mutation+in+idiopathic+Parkinson's+disease.">A common LRRK2 mutation in idiopathic Parkinson's disease.</a> Lancet. 365 (9457), 415-416 (2005).</li><li>Satake, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+study+identifies+common+variants+at+four+loci+as+genetic+risk+factors+for+Parkinson's+disease.">Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease.</a> Nat. Genet. 41 (12), 1303-1307 (2009).</li><li>Sim&#243;n-S&#225;nchez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+study+reveals+genetic+risk+underlying+Parkinson's+disease.">Genome-wide association study reveals genetic risk underlying Parkinson's disease.</a> Nat. Genet. 41 (12), 1308-1312 (2009).</li><li>Klein, C., Ziegler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imputation+of+sequence+variants+for+identification+of+genetic+risks+for+Parkinson's+disease:+a+meta-analysis+of+genome-wide+association+studies.">Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies.</a> Lancet. 377 (9766), 641-649 (2011).</li><li>Cookson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+leucine-rich+repeat+kinase+2+(LRRK2)+in+Parkinson's+disease.">The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease.</a> Nat. Rev. Neurosci. 11 (12), 791-797 (2010).</li><li>Ozelius, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+G2019S+as+a+Cause+of+Parkinson’s+Disease+in+Ashkenazi+Jews.">LRRK2 G2019S as a Cause of Parkinson’s Disease in Ashkenazi Jews.</a> N. Engl. J. Med. 354 (4), 424-425 (2006).</li><li>Lesage, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+G2019S+as+a+Cause+of+Parkinson’s+Disease+in+North+African+Arabs.">LRRK2 G2019S as a Cause of Parkinson’s Disease in North African Arabs.</a> N. Engl. J. Med. 354 (4), 422-423 (2006).</li><li>Bouhouche, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+G2019S+Mutation:+Prevalence+and+Clinical+Features+in+Moroccans+with+Parkinson's+Disease.">LRRK2 G2019S Mutation: Prevalence and Clinical Features in Moroccans with Parkinson's Disease.</a> Parkinsons. Dis. , 1-7 (2017).</li><li>West, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease-associated+mutations+in+leucine-rich+repeat+kinase+2+augment+kinase+activity.">Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (46), 16842-16847 (2005).</li><li>Ito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP+binding+is+essential+to+the+protein+kinase+activity+of+LRRK2,+a+causative+gene+product+for+familial+Parkinson's+disease.">GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease.</a> Biochemistry. 46 (5), 1380-1388 (2007).</li><li>Steger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteomics+reveals+that+Parkinson's+disease+kinase+LRRK2+regulates+a+subset+of+Rab+GTPases.">Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.</a> Elife. 5, (2016).</li><li>Ito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phos-tag+analysis+of+Rab10+phosphorylation+by+LRRK2:+a+powerful+assay+for+assessing+kinase+function+and+inhibitors.">Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.</a> Biochem. J. 473, 2671-2685 (2016).</li><li>Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., Koike, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-binding+tag,+a+new+tool+to+visualize+phosphorylated+proteins.">Phosphate-binding tag, a new tool to visualize phosphorylated proteins.</a> Mol. Cell. Proteomics. 5 (4), 749-757 (2006).</li><li>Using a Hemacytometer to Count Cells. J. Vis. Exp Available from: <a target="_blank" href="https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells">https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells</a> (2017)</li><li>Ni, D., Xu, P., Gallagher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+Immunodetection.">Immunoblotting and Immunodetection.</a> Curr. Protoc. Mol. Biol. (114), 10.8.1-10.8.37 (2016).</li><li>Reith, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK2578215A;+a+potent+and+highly+selective+2-arylmethyloxy-5-substitutent-N-arylbenzamide+LRRK2+kinase+inhibitor.">GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.</a> Bioorg. Med. Chem. Lett. 22 (17), 5625-5629 (2012).</li><li>Fell, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MLi-2,+a+potent,+selective+and+centrally+active+compound+for+exploring+the+therapeutic+potential+and+safety+of+LRRK2+kinase+inhibition.">MLi-2, a potent, selective and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition.</a> J. Pharmacol. Exp. Ther. 355, 397-409 (2015).</li><li>Dzamko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+LRRK2+kinase+activity+leads+to+dephosphorylation+of+Ser(910)/Ser(935),+disruption+of+14-3-3+binding+and+altered+cytoplasmic+localization.">Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.</a> Biochem. J. 430 (3), 405-413 (2010).</li><li>Th&#233;venet, J., Pescini Gobert, R., Hooft van Huijsduijnen , R., Wiessner, C., Sagot, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+LRRK2+expression+points+to+a+functional+role+in+human+monocyte+maturation.">Regulation of LRRK2 expression points to a functional role in human monocyte maturation.</a> PLoS One. 6 (6), e21519 (2011).</li></ol>

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ここでは、P タグの方法論に基づいて内因性レベル LRRK2 による Rab10 リン酸化を検出する簡易で堅牢な方法をについて説明します。現在利用可能なリン酸化 Rab10 抗体発現タンパク質15でのみ動作するため、P タグ SDS-PAGE を利用した本法は Rab10 リン酸化の内因性のレベルを評価する唯一の方法です。さらに、本法は、Rab10 リン酸化細胞の化学組成の推定できます。P タグの方法...

PD は、主に高齢者1モーター システムの機能不全の結果、中脳のドーパミン作動性ニューロンに影響最も一般的な神経変性疾患の一つです。ほとんどの患者は、PD を作成し散発的な方法で、病気を継承する家族が集まっています。FPD2とリンクするいくつかの遺伝子の突然変異が見つかっています。FPD の原因遺伝子の 1 つは、LRRK2、パーク 8 と呼ばれる支配的継承された FPD にリンク 8 のミスセンス変異 (N1437H、R1441C、G、H、S、Y1699C、G2019S、および I2020T) にされているの報告された3,4,5これまでのところです。いくつかのゲノムワイド関連研究 (GWAS) 散発的な PD 患者は PD、LRRK2 の機能に異常が散発的の両方の神経変性疾患の一般的な原因であることを示唆しているのリスク要因として LRRK2 軌跡におけるゲノム変化も確認します。パーク 8 FPD6,7,8。
LRRK2 は、ロイシン豊富な繰り返しドメイン、複雑なタンパク質 (中華民国) ドメイン、ROC (COR) ドメイン、セリン/スレオニン蛋白質キナーゼ ドメインおよび9WD40 リピート ドメインの C 末端の GTP 結合 Ras から成る大規模な蛋白質 (2,527 アミノ酸) であります。8 つの FPD 変異をこれらの機能ドメインの検索します。N1437H と ROC ドメイン、COR ドメイン、G2019S とキナーゼ ドメインで I2020T で Y1699C で R1441C/G/H/S。それは、仮定される G2019S 突然変異は、PD 患者10、11,12の突然変異を見つけた最も頻繁には、体外で2-3 倍13LRRK2 のキナーゼ活性が増加、のでLRRK2 substrate(s) のリン酸化の異常な増加は神経細胞に有毒であります。ただし、それされています患者派生サンプルで評価方法の不足のため散発的/家族性 PD 患者における生理学的関連 LRRK2 基板のリン酸化が変更されるかどうかを検討することが可能。
蛋白質のリン酸化は免疫ブロットまたは質量分析によるタンパク質のリン酸化状態を具体的に認識する抗体を用いた酵素抗体法 (ELISA) によって一般に検出されます。ただし、元の戦略時適用できませんリン酸化特異的抗体を作成する難しさのため。放射性隣酸塩に細胞の代謝ラベリングはリン酸化特異的抗体が容易に利用可能でない場合、リン酸化の生理的レベルを確認する別のオプションです。ただし、放射性物質の大規模な量を要求され、したがって放射線防護14のために特殊な機器を伴います。質量分析はより敏感なこれらの免疫化学的方法と比較して、蛋白質のリン酸化の分析に人気となったです。サンプル準備は時間がかかるし、高価な楽器が分析に必要です。
Rab10 Rab8 など Rab GTPase の家族のサブセットは、LRRK2 の直接生理学的な基板が15大規模プロテオーム解析の結果に基づき、最近報告されました。[Rab10 リン酸化された FPD 変異マウス胚性線維芽細胞、マウス16をノックの肺によって増加するデモンストレーションを行った。本報告では、ナトリウム dodecyl 硫酸塩ポリアクリルアミドゲル電気泳動 (SDS-PAGE) を採用しました-P タグ分子が SDS ページのゲル (P タグ SDS-PAGE) Rab10 リン酸化の内因性のレベルを検出するために共重合する手法リン酸化 Rab10 の高感度抗体はまだ欠けていた。我々 は合計 Rab8 の現在入手可能な抗体の選択度が低くによる内因性 Rab8 のリン酸化を検出する失敗します。したがって、我々 は Rab10 リン酸化に焦点を当てることを決めた。LRRK2 は、Thr73 の非常に節約された「スイッチ II」地域の中で場所に行く Rab10 を廃止します。Rab 蛋白質リン酸化のサイトの高い保護は明確な Rab 蛋白質を認識する検体抗体が作りにくい理由の一つかもしれません。
LRRK2 による Rab8A のリン酸化は、Rabin8、GTP15にバインドされた GDP を交換することによって Rab8A をアクティブにグアニン ヌクレオチド交換因子 (GEF) の結合を阻害します。Rab10、LRRK2 による Rab8A のリン酸化は、Rab タンパク質の活性化に不可欠な膜15から GDP 結合 Rab 蛋白質の抽出は、GDP 解離阻害剤 (GDIs) の結合を阻害します。総称して、それは、LRRK2 による Rab タンパク質のリン酸化を防ぎます活性化正確な分子機構とリン酸化の生理的影響は明白でなく残るが、仮定されます。
P タグ SDS ページは 2006 年に木下らによって発明された: この方法ではアクリルアミドは P タグと共有結合は、SDS-PAGE に共高親和とリン酸塩を取り込み分子ゲル17。SDS ページのゲルの P タグ分子選択的リン酸化タンパク質の電気泳動移動度を遅らせる、ので P タグ SDS-PAGE は非リン酸化のものからリン酸化タンパク質を区切ることができます (図 1)。興味の蛋白質は複数の残基のリン酸化、リン酸化特異的形態に対応するバンドのはしごは見られなくなります。Rab10、場合 Rab10 で Thr73 だけでリン酸化されることを示す 1 つだけ移されたバンドを観察します。リン酸化特異的抗体と免疫ブロット上 P タグ SDS ページの主な利点は非リン酸化特異抗体 (すなわち、認識合計 Rab10) とイムノブロット リン酸 Rab10 が認識されること膜に転送された後である一般により特定、機密、および利用可能な商業学術ソースから。SDS ページ P タグを使用してのもう一つの利点は、リン酸化特異的抗体と免疫ブロットすること放射性と細胞の代謝ラベリングによって不可能である 1 つは、リン酸化の化学量論の近似的評価を取得できることリン酸塩。
別に安価な P タグ アクリルアミドおよびそれに関連するいくつかのマイナーな変更の使用は、現在 LRRK2 による Rab10 リン酸化の検出法免疫ブロットの一般的なプロトコルに従います。したがって、免疫ブロットが浄化された蛋白質、セル lysates の組織乳剤などのサンプルの任意のタイプの通常の練習を任意の所で簡単であり、容易に実行可能ファイルをする必要がありますそれ。

<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

ロイシン豊富な繰り返しキナーゼ 2 (LRRK2) の変異は、家族性パーキンソン病 (FPD) とリンクする示されています。LRRK2 のキナーゼ活性の異常な活性化は、PD の発症機序に関与している、ので LRRK2 キナーゼ活性の生理的レベルを評価する手法の確立が必要不可欠です。最近の研究では、LRRK2 廃止 Rab10 を含む生理学的な条件の下で、Rab GTPase 家族のメンバーであることを明らかにしました。質量分析法による培養細胞における LRRK2 による内因性 Rab10 のリン酸化を検出する可能性があります、されているイムノブロットの現在入手可能な特定のリン酸化抗体の感度不良のためそれを検出することは困難Rab10。ここで、簡単な説明ナトリウム dodecyl 硫酸塩ポリアクリルアミドゲル電気泳動 (SDS-PAGE) リン酸バインディング タグ (P タグ)、併用を利用した免疫ブロットに基づく LRRK2 による Rab10 リン酸化の内因性のレベルを検出します。N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N'、N'- トリス (ヌクレオチ-2-イル-メチル) - 1, 3 - diaminopropan-2-ol。現在のプロトコルだけでなく P タグを利用する方法の例を提供します、どのように変異と同様阻害剤治療/管理やその他の要因を変える細胞や組織における LRRK2 の下流のシグナリングの評価ができます。.

過剰発現システム: ハ-Rab10 3 のリン酸化 × フラグ-LRRK2 の HEK293 細胞します。
HEK293 細胞は、HA Rab10 野生型および 1.066 μ g 3 × フラグ LRRK2 の (野生型、キナーゼ不活性変異体 (K1906M)、または FPD 変異) 0.266 μ g を導入させた。反を用いたイムノブロット P タグ SDS-PAGE を用いて Rab10 リン酸化-HA 抗体 (図 2)。10 ...

Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

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

本研究では、ロイシン豊富な繰り返しキナーゼ 2 によって Rab10 リン酸化の内因性のレベルを検出する簡単な方法について説明します。

SDS ページを用いたリン酸 binding タグ LRRK2 活動によって Rab10 リン酸化検出

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

14.9K ビュー.  The University of Tokyo. 本研究では、ロイシン豊富な繰り返しキナーゼ 2 によって Rab10 リン酸化の内因性のレベルを検出する簡単な方法について説明します。

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

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

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.

リン酸化部位決定のためのオリゴペプチド競合アッセイ

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

生化学

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

サイクリン依存性キナーゼ 1 の同定の in Vitroキナーゼによって特定のリン酸化のサイトの試金します。

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

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

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;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.

Rab10リン酸化を評価してパーキンソン病関連LRRK2キナーゼ経路を尋問するためのヒト末梢血好中球分離

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

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.

多彩な生化学的アプローチを用いた新規 CK2 キナーゼ基板の識別

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

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

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

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 &#947;[32P] ATP labeling.

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

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 of LRRK2 in disease.

LRRK2のキナーゼ活性を測定する in vitroで</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

生物学

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

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

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.

ロイシンリッチの代謝標識は、放射性リン酸キナーゼ1および2を繰り返します

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

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.

IRF3のリン酸化依存性活性化を監視するために、高解像度の方法

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

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

Source: Bailey, E. M. et al., <a href="https://www.jove.com/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> J. Vis. Exp. (2022)
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.

タンパク質リン酸化分析のための単一分子プルダウンアッセイ:細胞溶解物中のタンパク質リン酸化を定量するハイスループット技術

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)
This video ...