Dr. Takeshi Iwatsubo (University of Tokyo, Japonya) lütfen 3xFLAG-LRRK2 WT ve mutantlar kodlama plazmid verdiğiniz için teşekkür ederiz. Biz de Dr Dario Alessi (Dundee Üniversitesi, İngiltere) nazikçe mlı-2 ve HA-Rab10 kodlama plazmid verdiğiniz için teşekkür ederiz. Bu eser bilim promosyon (JSP'ler) KAKENHI Grant numarası JP17K08265 (G.I.) için Japonya Derneği tarafından desteklenmiştir.

<ol>
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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. 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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. 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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.bdigitaluss.remotexs.co/science-education/5048/using-a-hemacytometer-to-count-cells">https://www-jove-com.bdigitaluss.remotexs.co/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. 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Yazarlar ifşa gerek yok.

Burada, Rab10 fosforilasyon tarafından LRRK2 P-etiket yöntembilimi'ne dayalı endojen düzeylerde tespit facile ve sağlam yöntemi açıklanmaktadır. Fosforile Rab10 karşı şu anda mevcut antikor ile sadece15overexpressed proteinler çalıştığından, P-tan SDS-sayfası kullanan mevcut yöntemi Rab10 fosforilasyon endojen düzeylerini değerlendirmek için tek yolu bu. Ayrıca, mevcut yöntemi Rab10 fosforilasyon hücrelerdeki stoichiometry tahmin sağlar. P-etiket metodoloji fosfo-protein...

PD en sık görülen nörodejeneratif hastalıklar, ağırlıklı olarak orta dopaminerjik nöronlarda disfonksiyon yaşlı insanlar1motor sistemleri sonuçlanan etkileyen biridir. Hastaların çoğunda PD sporadik bir şekilde geliştirmek iken hastalığı devralan aileler. Bazı genlerdeki mutasyonlar FPD2ile bağlantılı olarak tespit edilmiştir. FPD için sorumlu gen biridir LRRK2, sekiz missense PARK8 denilen bir baskın devralınan FPD bağlı mutasyonlar (N1437H, R1441C/G/H/S, Y1699C, G2019S ve I2020T) şimdiye bildirilen3,4,5olmuştur. Çeşitli genom çapında dernek çalışmaları (GWAS) sporadik PD hastaların da LRRK2 odağı genomik varyasyonları bir risk faktörü PD, anormallik LRRK2 işlevindeki ateş sporadik hem de yaygın bir nedeni olduğunu düşündüren için belirledik ve PARK8 FPD6,7,8.
LRRK2 lösin yönünden zengin tekrar alanının bir etki alanı, GTP bağlayıcı Ras karmaşık proteinleri (ROC) etki alanı, C-terminal ROC (COR) etki alanı, bir serin/treonin protein kinaz etki ve WD40 tekrar etki alanı9oluşan büyük bir protein (2527 amino asitler) olduğunu. Sekiz FPD mutasyonlar bu işlevsel etki alanlarında bulun; N1437H ve R1441C/G/H/S Y1699C COR etki alanında, G2019S ve I2020T kinaz etki alanındaki ROC etki alanında. Bu yana en sık mutasyon PD hastalar10,11,12' bulundu, G2019S mutasyon LRRK2 kinaz aktivitesinin 2-3 kat vitro13tarafından artırır, onaylanmadığına karar LRRK2 substrate(s) fosforilasyon anormal artış nöronlar için toksik. Ancak, fizyolojik ilgili LRRK2 yüzeylerde fosforilasyon hasta türetilmiş örneklerinde değerlendiren yöntemler eksikliği nedeniyle ailesel/sporadik PD hastalarında değişmiş olup olmadığını incelemek mümkün olmuştur.
Protein fosforilasyon genellikle immunoblotting ya da özellikle devletin fosforile proteinlerin veya kitle spektrometrik analizi tarafından tanıma antikorları kullanarak enzim bağlı immunosorbent assay (ELISA) tarafından algılanır. Ancak, eski strateji fosforilasyon özgü antikor oluşturma zorlukları nedeniyle bazen uygulanamaz. Radyoaktif fosfat içeren hücrelerin metabolik etiketleme fosforilasyon özel antikorlar kolayca kullanılabilir olduğunda fosforilasyon fizyolojik düzeyleri incelemek için başka bir seçenektir. Ancak, radyoaktif malzeme büyük miktarda gerektirir ve bu nedenle bazı özel ekipman radioprotection14için içerir. Kitle spektrometrik analizi daha duyarlı bu immunochemical yöntemlerine göre ve protein fosforilasyon analiz popüler oldu. Ancak, örnek hazırlıktır zaman alıcı ve pahalı aletler analiz için gereklidir.
LRRK2 için doğrudan fizyolojik yüzeylerde bir büyük ölçekli phosphoproteomic analiz15sonucuna dayalı olarak bir alt Rab10 ve Rab8 de dahil olmak üzere Rab GTPazlar ailesinin son zamanlarda bildirildi. O zaman Rab10 fosforilasyon FPD mutasyonlar fare embriyonik fibroblastlar ve fareler16knockin akciğerler yükseltilmiştir gösterdi. Bu raporda, biz bir Sodyum Lauryl Sülfat polyacrylamide Jel Elektroforez (SDS-sayfa) istihdam için seçti-P-etiket molekül Rab10 fosforilasyon, endojen düzeylerini saptamak için SDS-sayfa jelleri (P-tan SDS-sayfası) içine co polimerli yöntemi dayalı Çünkü son derece hassas antikor için fosforile Rab10 belirli hala eksik. Şu anda kullanılabilir antikorlar kötü selectivity nedeniyle endojen Rab8 fosforilasyon için toplam Rab8 algılamak başarısız oldu. Bu nedenle, Rab10 fosforilasyon üzerinde odaklanmaya karar verdi. LRRK2 Rab10 Thr73 son derece korunmuş geçiş"II" bölgesinin ortasında bulma phosphorylates. Yüksek koruma Rab proteinler arasında fosforilasyon sitelerin neden farklı Rab proteinler tanıma phosphospecific antikorlar yapmak zor nedenlerinden biri olabilir.
Rab8A LRRK2 tarafından fosforilasyon Rabin8, GTP15ile ilişkili GSYİH değiş tokuş ederek Rab8A etkinleştiren bir guanin nükleotid Satım faktörü (GEF) bağlama engeller. Fosforilasyon, Rab10 ve LRRK2 tarafından Rab8A de GSYİH bağlı Rab proteinler membran15ayıklayarak Rab proteinleri harekete geçirmek için gerekli olan GSYİH-ayrılma inhibitörleri (GDIs), bağlama engeller. Topluca, hassas moleküler mekanizması ve fosforilasyon fizyolojik sonuçları belirsiz kalır, ancak fosforilasyon tarafından LRRK2 Rab proteinlerin onları aktivasyon engellediğini onaylanmadığına karar.
P-etiket SDS-sayfa 2006 yılında Kinoshita vd tarafından icat edilmiştir: Bu yöntemde, Akrilamid kovalent P etiketi ile birleştiğinde, bir molekül fosfat SDS-sayfa copolymerized yüksek benzeşimli yakalama17jelleri. SDS-sayfa jel P etiketi molekülleri seçerek fosforile proteinlerin elektroforetik hareketlilik geri zekalı çünkü P etiketi SDS-sayfa fosforile proteinlerin sigara fosforile olanlardan ayırabilirsiniz (şekil 1). Faiz protein üzerinde birden fazla artıkları fosforile Eğer bir merdiven differentially fosforile biçimlerine karşılık gelen gruplarından gözlenir. Rab10 söz konusu olduğunda, biz Rab10 sadece Thr73 fosforile gösteren tek bir grup değiştirdi, gözlemlemek. İmmunoblotting fosforilasyon özel antikorlar ile büyük avantaj P etiketi SDS-sayfanın fosforile Rab10 fosforilasyon spesifik antikorlar (Yani, tanıma toplam Rab10) ile immunoblotting tarafından tespit edilebilir olduğunu membranlar transfer sonra genellikle ticari/akademik kaynaklardan daha belirli, hassas ve kullanılabilir olduğu. Bir immunoblotting fosforilasyon özel antikorlar ile veya radyoaktif hücrelerle metabolik etiketlerine göre hangi imkansız fosforilasyon, stoichiometry yaklaşık tahmini elde edebilirsiniz P etiketi SDS-sayfa kullanmanın bir diğer avantajı olduğunu fosfatlar.
Ucuz P etiketi Akrilamid ve bazı küçük değişiklikler ile ilgili kullanımı dışında Rab10 fosforilasyon LRRK2 tarafından algılanması için mevcut yöntemi immunoblotting genel bir protokol izler.Bu nedenle, immunoblotting örnek saf proteinler, hücre lysates ve doku homogenates dahil herhangi bir türde her zamanki bir uygulama nerede herhangi bir laboratuarlarında basit ve kolayca çalıştırılabilir olması gerektiği.

<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

Lösin yönünden zengin tekrar kinaz 2 (LRRK2) mutasyonların ailesel Parkinson hastalığı ile (FPD) bağlanacak şekilde gösterilmiştir. LRRK2 kinaz aktivitesinin anormal harekete geçirmek PD patogenezinde karıştığı olmuştur, bu LRRK2 kinaz aktivitesinin fizyolojik düzeylerini değerlendirmek için bir yöntem kurmak için gereklidir. Son yıllarda yapılan çalışmalarda LRRK2 fizyolojik koşullar altında Rab10, Rab GTPazlar ailesinin üyeleri phosphorylates ortaya koydu. Her ne kadar tarafından LRRK2 kültürlü hücrelerde endojen Rab10 fosforilasyon tarafından kütle spektrometresi tespit edilemedi, immunoblotting için şu anda mevcut fosforilasyon özgü antikorların zavallı duyarlılık nedeniyle tarafından tespit etmek zor oldu Rab10. İşte, biz basit bir tarif Rab10 fosforilasyon tarafından LRRK2 endojen düzeylerini tespit yöntemi bir fosfat bağlayıcı etiketi (P-etiket), ile birlikte Sodyum Lauryl Sülfat polyacrylamide Jel Elektroforez (SDS-sayfa) kullanan immunoblotting dayalı olan N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl) -N,N',N'- tris (pyridin-2-yl-metil) - 1,3 - diaminopropan-2-ol. Mevcut Protokolü sadece P etiketi kullanan metodoloji örneği sağlar ama aynı zamanda hücre ve dokulara LRRK2 aşağı akım sinyal mutasyonlar yanı sıra inhibitörü tedavisi/yönetim veya herhangi bir diğer etken nasıl değiştirmek değerlendirmek sağlar .

Overexpression sistemi: HA-Rab10 3 tarafından fosforilasyon × bayrak-LRRK2 içinde HEK293 hücreleri:
HEK293 hücreleri HA-Rab10 vahşi tipi ve 1,066 µg 3 × bayrak-LRRK2 (yaban-tipi, etkin olmayan kinaz mutant (K1906M) veya FPD mutantlar) 0,266 µg ile transfected. Rab10 fosforilasyon P etiketi SDS-sayfa bir anti kullanarak immunoblotting tarafından takip tarafından muayene-HA antikor (Şekil 2)....

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

Bu da çalışmanın Rab10 fosforilasyon endojen düzeyleri lösin yönünden zengin tekrar kinaz 2 algılama basit bir yöntem açıklanır.

Rab10 fosforilasyon algılama SDS-sayfa bir fosfat bağlayıcı etiketi ile kullanarak LRRK2 etkinliğini tarafından

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

14.9K Görüntüleme.  The University of Tokyo. Bu da çalışmanın Rab10 fosforilasyon endojen düzeyleri lösin yönünden zengin tekrar kinaz 2 algılama basit bir yöntem açıklanır.

Video: 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.

Fosforilasyon Saha Tespiti İçin Oligopeptide Rekabet Testi

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

Biyokimya

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.

Siklin bağımlı kinaz 1 tanımlaması belirli fosforilasyon siteleri bir Vitro kinaz tarafından tahlil

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 Fosforilasyon Değerlendirilmesi tarafından Parkinson İlişkili LRRK2 Kinas yolu Sorgulama için İnsan Periferik Kan Nötrofil İzolasyon

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.

Kimliği roman CK2 kinaz yüzeylerde çok yönlü bir biyokimyasal yaklaşım kullanarak

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.

Yeni kinaz proteinin sağlam biyokimyasal yaklaşımlar kullanılarak moleküler düzeyde karakterize edilmesi

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 Kinaz Aktivitesi Assaying In vitro</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 ...

Biyoloji

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.

Lösin zengin metabolik etiketleme Fosfat ile radyoaktif Kinazlar 1 ve 2, tekrar

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.

Bir Yüksek Çözünürlüklü Yöntem IRF3 fosforilasyonu bağımlı Aktivasyon Monitör

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.bdigitaluss.remotexs.co/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.

Protein Fosforilasyon Analizi için Tek Moleküllü Aşağı Çekme Testi: Hücre Lizatında Protein Fosforilasyonunu Ölçmek İçin Yüksek Verimli Bir Teknik

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