Biz Dr David King, Howard Hughes tıp Enstitüsü, University of California Berkeley kütle spektrometresi analiz ve yararlı yorumlar için teşekkür ederiz. Biz Dr Xuelian Zhu, Shanghai, Biyoloji Bilimleri enstitüleri, Çin Bilimler Akademisi, tam uzunlukta bir CENP-F yapı sağlamak için Shanghai, Çin teşekkür ederim. Son olarak, biz Dr Susan Bane, Dr Brian Callahan ve Dr Christof Grewer Binghamton Üniversitesi'nde ekipmana için teşekkür ederim. Bu araştırma State University of New York ve State University of New York Binghamton, Kimya bölümü için Araştırma Vakfı tarafından finanse edildi.

Yazarlar ifşa gerek yok.

Bizim vitro kinaz tahlil hangi hücre döngüsünün ana denetleyicisi ve birçok önemli hücresel süreçler düzenleyen kinaz Cdk1, moleküler hedefleri tanımlamak için çok güçlü bir yöntem olduğunu. Yöntem bir saf protein bir substrat Cdk1 için ve belirli fosforilasyon sitelerin kimlik sağlar Eğer belirler. Bu Cdk1 aracılığıyla fosforilasyon tarafından mekanik çalışmaları, düzenleme, hücresel süreçler için kolaylaştırır.
En kritik kütle spektrometresi...

Kinazlar fosfat grupları ATP yüzeyler transfer ve birçok hücresel süreçler düzenleyen enzimlerdir. Bu fosforilasyon geri alınabilir, hızlı, iki negatif ücretleri, ekler ve serbest enerji depolar ve hücreleri tarafından kullanılan en yaygın ardından değişiklikler biridir. Hücre bölünmesi döngüsü protein 2 homolog (cdc2) olarak da bilinen olan Cdk1 hücre döngüsünün bütün Ökaryotlar1,2,3,4,5ana bir denetleyicisidir ve phosphorylates bir yaklaşık 8-%13 Proteom6,7.
Bu değişiklikler için sorumlu kinaz bilinmeyen ise son proteomik çalışmalar proteinler, çoğu durumda, çok sayıda fosforilasyon site belirledik. Özellikle insan hücrelerinde bilinen Cdk1 hedeflerin sayısı düşük7var. Nasıl Cdk1 hücre döngüsü kontrol kurmak mekanik çalışmalar kullanmasına olanak tanır Cdk1 özgü fosforilasyon siteler tanımlanması önemlidir. Hücre döngüsü Yönetmeliği sadık kromozom segregasyon ve hücre bölünmesi için önemlidir ve hücresel sayısız bu önemli fizyolojik işlevi desteklemek için ortaya çıkması gereken. Bu transkripsiyon ve translasyon hücre yapısı ve organizasyon, nükleer zarf, kromozom yoğunlaşma ve Mitotik iğ Montaj Demontaj gibi dramatik bir yeniden yapılanma yanı sıra mitoz başlangıcı öncesinde durdurma içerir. Deregülasyon ve hataları bu süreçlerde kanser, doğum kusurları veya Mitotik hücre ölümüne neden olur. Cdk1 RO-3306 gibi belirli inhibitörleri fonksiyonel çalışmaları için güçlü araçlar sağlayan gelişmiş8, vardı ve bu inhibitörleri şu anda kanser tedavisi için klinik çalışmalarda bazı (9 inceleme için bakınız).
Burada, biz Cdk1 özgü fosforilasyon siteler tanımlaması sağlar bir vitro kinaz tahlil açıklayın. Bu tahlil, ticari olarak mevcut insan Cdk1/siklin B arıtılmış hedef protein vitrofazdan için kullanılır. Bir substrat fosforilasyon kütlesi artar ve iki negatif ücretleri ekler; Bu nedenle, başarılı fosforilasyon SDS-sayfa protein jel grubu yukarı doğru bir kayma tarafından onaylanır. Cdk1 özgü fosforilasyon siteleri daha sonra kütle spektrometresi fosforile vitro protein analizi tarafından tanımlanır. Deneysel tasarım ile yardımcı olmak için biz de Hesaplama araçları ve protein serisinden Cdk1 özgü fosforilasyon siteler tahmin için başvurular gözden geçirin. Ayrıca, aynı zamanda son derece saf ve homojen protein kinaz tahlil için uygun hazırlıklar verim arıtma iletişim kuralları açıklar. Son olarak, tanımlanan fosforilasyon siteleri fonksiyonel çalışmaları tarafından doğrulanması gerekir ve bir basit bağlama tahlil burada bu amaçla kullanılmıştır. Birlikte, bu Cdk1 özgü fosforilasyon siteler verimleri ve mekanik çalışmaları nasıl hücre döngüsü7,10,11Cdk1 denetler içine sağlayan çok güçlü bir yaklaşım olacaktır. Bu yöntem üzerinde saf protein dayanıyor beri herhangi bir model organizma ve verimleri güvenilir sonuçlar için uygulanabilir. Yerde, ardından değişiklikleri, etkileşim ortakları veya hücrede yerleşimi gibi ek düzenleyici mekanizmaları hücre var gibi Ancak, elde edilen fosforilasyon siteleri vitro işlevsel doğrulaması, tavsiye bu fosforilasyon siteleri erişilebilir veya Cdk1 tarafından tanınması için erişilebilir hale gelebilir.
Cdk1 (Ser/Thr-Pro-X-Lys/Arg), burada X herhangi bir kalıntı ve serin veya treonin fosforilasyonu sitedir oluşur bir fikir birliği fosforilasyon site tanır. + 1 pozisyon prolin varlığı tanıma için özellikle önemlidir. Buna ek olarak, temel kalıntıları + 2 veya 3 pozisyonlarda tercih edilmektedir, Lys veya Arg 3 içeren çoğu Cdk1 özgü fosforilasyon site ile6,12konum.
Cdk1 aktivasyonu sıkı bir şekilde düzenlenir ve mitoz1,2,3,4,5başlangıcı için yol açar. Siklin bağımlı kinaz aktivitesinin genel olarak onların dernek ile farklı siklinlere (siklin A, B, C, D ve E insanlarda), hücre döngüsü13boyunca düzeyleri salınan ifade edilir bağlıdır. Cdk1 ifade hücre döngüsü boyunca sabittir ve siklin A ve B5,13,14,15, olarak siklin düzenleyici alt birimleri ile dernek, düzenleme, faaliyete dayanan de translasyon modifikasyon. Cdk1/siklin B kompleks oluşumu kinaz harekete geçirmek5,14,15,16,17,18için gereklidir. G2 evresinde, siklin B sitozol tercüme ve nerede Cdk15,14,15,16,17,-18bağlar çekirdeği'a içe aktarılan; Ancak, B Cdk1/siklin fosforilasyon (membran ilişkili tirozin ve treonin özel cdc2 inhibitör kinaz) artıkları Thr14 ve Tyr15 insan Cdk1 inhibitör kinaz Myt1 tarafından ve Wee1, sırasıyla19tarafından Inaktif tutulur, 20,21. Geç G2 aşamasında, hücre bölünmesi döngüsü 25 fosfataz (cdc25) tarafından Thr14 ve Tyr15 dephosphorylation Cdk1/siklin B kompleks kinaz aktivitesinin etkinleştirir ve mitoz12,14, inisiyasyon tetikler 18 , 20 , 22 , 23. Thr161 fosforilasyon da Cdk1/siklin B harekete geçirmek için gereklidir ve Cdk7, Cdk aktive kinaz (CAK)18aracılık ettiği. Siklin B anafaz bozulma Cdk1, mitoz24,25dan çıkış sağlayan inaktive. B Cdk1/siklin aktivasyonu bu nedenle karmaşık bir süreçtir. Burada sunulan Protokolü piyasada bulunan Cdk1/siklin b ile gerçekleştirilir Böcek hücrelerinde rekombinant ifade bu kompleksin sırasında bu harekete geçirmek içinde vivo endojen kinaz14,20 ve arıtılmış durumda etkin kalır. Elde edilen aktif, rekombinant insan Cdk1/siklin B vitro kinaz deneyleri için uygundur.
Burada, biz Cdk1 özgü fosforilasyon siteler insan şeması protein F (CENP-F)10tanımlaması için bir iletişim kuralı tanımlamak. CENP-F, interfaz (G1 ve S-fazlı) en sırasında çekirdeğinde bulunan ve Cdk1 bağımlı bir şekilde10, G2 aşama26,27,28 sitozol verilir bir kinetochore protein 11. nükleer yerelleştirme önerebilirim cNLS26tarafından haiz. kolaylaştırır, birlikte karyopherin β ve RanGDP, cNLS-yük alma çekirdeği29içine nükleer taşıma faktörü karyopherin α cNLSs algılanır. G2 aşamasında nükleer verme yolu ile an bilinmeyen verme yolu10kolaylaştırılmıştır. CENP-F sitozol içinde bulunduğu nükleer zarf oluşturulmuştur ve sırayla motor protein kompleksi dinein30,31acemi. Bu yol için centrosome ilgili çekirdek Mitotik Milli Meclisi ilk aşamalarında Mitotik girdinin doğru zamanlama ve beyin temel bir işlemde önemlidir dinein bağımlı bir şekilde konumlandırmak önemlidir geliştirme30,31,32. G2 aşamasında başlayan, CENP-F de kinetochore sadık kromozom segregasyon27,28,33,34,35 için önemli roller bulunduğu monte edilir . Bir anahtar düzenleyici bu yollar CENP-F nükleer ihracat Cdk110,11tarihinde bağlıdır G2 aşamasında adımdır. Biz burada bir protokol Cdk1 özgü fosforilasyon siteler CENP-F. cNLS içinde tanımlanması için tarif Phosphomimetic mutasyonlar bu sitelerin CENP-F B Cdk1/siklin doğrudan CENP-F Hücrede yerleşimi onun cNLS10fosforilasyon tarafından düzenleyen düşündüren, nükleer alınmasıyla yavaşlatacak.
Kinaz Cdk1. saf hedef proteinler fosforile vitro piyasada bulunan Cdk1/siklin B kompleks ve fosforilasyon siteler için genel olarak, bu vitro kinaz tahlil belirli yüzeylerde tanımlaması sağlar daha sonra kütle spektrometresi tarafından tanımlanır. Cdk1 özgü fosforilasyon siteler tanımlaması nasıl Cdk1 hücre döngüsü kontrol ortaya mekanik çalışmaları destekler.

<table>
	<tbody>
		<tr>
			<td>2800 ml baffled Fernbach flask</td>
			<td>Corning</td>
			<td>44232XL</td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Gold Biotechnology</td>
			<td>A-301-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Fisher Scientfiic</td>
			<td>BP413-25</td>
			<td></td>
		</tr>
		<tr>
			<td>benzamidine hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>B6506-25</td>
			<td></td>
		</tr>
		<tr>
			<td>bottletop filter</td>
			<td>Corning</td>
			<td>431161</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B recombinant, human 20,000 U/mL</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>EMD Millipore</td>
			<td>14-450</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>Invitrogen</td>
			<td>PV3292</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B + 10x PK buffer</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>CENP-F (residues 2987 &#8211; 3065) pGEX6P1 plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge: Heraeus Multifuge X3R, cooled, with TX-1000 swing-out rotor</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifugal filter units: Amicon Ultra-15 centrifugal filter units, 3 kDa cutoff, Ultracel-PL membranes</td>
			<td>EMD Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>chlorampenicol</td>
			<td>Gold Biotechnology</td>
			<td>C-105-100</td>
			<td></td>
		</tr>
		<tr>
			<td>D/L methionine</td>
			<td>Agros Organics / Fisher</td>
			<td>125650010</td>
			<td></td>
		</tr>
		<tr>
			<td>desalting pipet tips: Zip tips</td>
			<td>Millipore Sigma</td>
			<td>ZTC18S008</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable chromatography columns, Econo-Pac 1.5 x 12 cm</td>
			<td>Biorad</td>
			<td>7321010</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Gold Biotechnology</td>
			<td>DTT50</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Rosetta 2(DE3)pLysS strain</td>
			<td>EMD Millipore</td>
			<td>71403</td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization Fourier transform ion</td>
			<td>Bruker Amazon</td>
			<td>Apex III</td>
			<td></td>
		</tr>
		<tr>
			<td>cyclotron resonance mass spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization ion trap mass spectrometer</td>
			<td>Bruker Amazon</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>fixed angle rotor: Fiberlite F15-8x-50cy</td>
			<td>Thermo Scientific</td>
			<td>97040-276</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC system: &#196;kta Pure FPLC</td>
			<td>GE Healthcare</td>
			<td>29032697</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel filtration column: Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione agarose</td>
			<td>Pierce</td>
			<td>16101</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione, reduced</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>incubation shaker: multitron shaker</td>
			<td>Infors</td>
			<td>I10102</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Gold Biotechnology</td>
			<td>I2481C50</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>karyopherin &#945; pet-28a pres plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani medium</td>
			<td>Fisher Scientfiic</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge 5418R, refrigerated</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (0.5 ml)</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (1.5 ml)</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel affinity gel: His-Select Nickel affinity gel</td>
			<td>Millipore Sigma</td>
			<td>P6611-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-6P-1 plasmid</td>
			<td>Millipore Sigma</td>
			<td>GE28-9546-48</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Gold Biotechnology</td>
			<td>P470-10</td>
			<td></td>
		</tr>
		<tr>
			<td>PS protease: PreScission protease</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Phos-tag acrylamide</td>
			<td>Wako Pure Chem. Ind.</td>
			<td>304-93521</td>
			<td></td>
		</tr>
		<tr>
			<td>reduced gluthathione</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>roundbottom centrifuge tubes (Oakridge tubes)</td>
			<td>Fisher Scientfiic</td>
			<td>055291D</td>
			<td></td>
		</tr>
		<tr>
			<td>site-directed mutagenesis kit: QuikChange Lightning</td>
			<td>Agilent</td>
			<td>210518</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Mutagenesis Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sonifier: Branson S-250D sonifier</td>
			<td>Branson</td>
			<td>15 338 125</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra/Por 1RC dialysis membrane (6-8 kDa cutoff)</td>
			<td>Spectrum Labs</td>
			<td>08 670B</td>
			<td></td>
		</tr>
		<tr>
			<td>swing out rotor TX-1000</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of cyclin-dependent kinase 1 specific phosphorylation sites by an in vitro kinase assay

Siklin bağımlı kinaz 1 (Cdk1) hücre döngüsünün bütün Ökaryotlarda bir ana denetleyicisidir ve Proteom yaklaşık 8-%13 phosphorylates; Ancak, özellikle insan hücrelerinde Cdk1 için belirlenen hedeflerin sayısı hala düşük. Nasıl Cdk1 hücre döngüsü kontrol içine mekanik anlayışlar sağladıkları gibi Cdk1 özgü fosforilasyon siteler tanımlanması önemlidir. Hücre döngüsü Yönetmeliği sadık kromozom ayrımı için önemlidir ve bu karmaşık bir süreç içinde kusurları kromozom anomalileri ve kanser neden.
Burada, biz Cdk1 özgü fosforilasyon siteleri tanımlamak için kullanılan bir vitro kinaz tahlil açıklayın. Bu tahlil fosforile vitro arıtılmış bir proteindir ticari olarak mevcut insan Cdk1/siklin B. başarılı tarafından fosforilasyon SDS-PAGE tarafından onaylandıktan ve fosforilasyon siteleri daha sonra kütle spektrometresi tarafından tanımlanır. Ayrıca son derece saf ve homojen protein kinaz tahlil ve bağlama tahlil için uygun hazırlıklar arasındaki etkileşimi probları tanımlanan fosforilasyon siteleri fonksiyonel doğrulanması için verim arıtma iletişim kurallarını açıklar bir klasik nükleer yerelleştirme sinyal (cNLS) ve onun nükleer taşıma reseptör karyopherin α. Deneysel tasarım ile yardımcı olmak için protein dizilerinden Cdk1 özgü fosforilasyon siteler tahmin için yaklaşımlar gözden geçirin. Birlikte bu protokollerin Cdk1 özgü fosforilasyon siteler verimleri ve mekanik çalışmaları nasıl Cdk1 hücre döngüsü kontrol eder içine sağlayan çok güçlü bir yaklaşım sunuyoruz. Bu yöntem üzerinde saf protein dayanıyor beri bu herhangi bir model organizma ve verimleri güvenilir sonuçlar için özellikle hücre fonksiyonel çalışmalar ile birlikte uygulanabilir.

Son zamanlarda bir cNLS10bulunan bir CENP-F parçadaki Cdk1 özgü fosforilasyon siteleri tanımlamak için bir vitro kinaz tahlil (şekil 1) kullandık. Bu sinyal CENP-F nükleer yerelleştirme Interphase lerin confers. G2 evresinde, CENP-F sitozol Cdk1 bağlı şekilde çekirdek verilir. Nasıl CENP-f Hücrede yerleşimi Cdk1 düzenleyen üzerinde mekanik anlayışlar elde etmek için biz üreticisinin sunucu<sup class="xref...

Watch this Scientific Journal Video about Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay at JoVE.com

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

Siklin bağımlı kinaz 1 (Cdk1) hücre döngüsünün G2 aşamasında aktif ve birçok hücresel yolları düzenler. Burada, bir vitro kinaz tahlil için bir protokol ile bu önemli kinaz hücresel hedefleri oluşturmak üzere Cdk1 özgü fosforilasyon siteler tanımlaması sağlar Cdk1 mevcut.

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; ...

identification-cyclin-dependent-kinase-1-specific-phosphorylation

Research

JoVE Journal

Biochemistry

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

18.4K Görüntüleme.  State University of New York at Binghamton. Siklin bağımlı kinaz 1 (Cdk1) hücre döngüsünün G2 aşamasında aktif ve birçok hücresel yolları düzenler. Burada, bir vitro kinaz tahlil için bir protokol ile bu önemli kinaz hücresel hedefleri oluşturmak üzere Cdk1 özgü fosforilasyon siteler tanımlaması sağlar Cdk1 mevcut.

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

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

bir nitroselüloz Bağlama Deneyi kullanılarak bakteriyel Histidin Kinaz otofosforilasyon miktarının belirlenmesi

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Biyokimya

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein Kinaz Aktivitesini Radyoaktif Etiketli ATP ile Tahlil

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

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 ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA-Isopentenyl transferaz aktivite tespit etmek için bir In Vitro tahlil

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

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 ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

Rho kinase aktivitesinin geçici Inhibisyonu ile ınsan kaynaklı pluripotent kök hücreli türev Kardiyomiyositlerin engraftment artırılması

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

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 ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Kendi kendine monte Edilen İnsan Protein Mikrodizilerinde Kiaz Inhibitörü Taraması

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...