Name: metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate
Uploaded: 2013-09-18T11:00:00
Description: Watch this Scientific Journal Video about Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate at JoVE.com

我们也感谢迈克尔J.福克斯基金会支持本研究。我们感谢研究基金会 - 佛兰德FWO（FWO项目G.0666.09，高级研究员奖学金到JMT），内陆水运SBO/80020项目的神经靶点，在鲁汶大学（OT/08/052A和IOF-KP/07 / 001）他们的支持。这项研究是由基金Druwé-Eerdekens的博杜安国王基金会JMT管理也支持一部分。

本协议使用的放射性32 P标记的磷酸盐遵循LRRK2的细胞磷酸化。承担重要的是记住，使用放射性试剂的所有操作，应使用适当的防护措施，尽量减少放射性辐射暴露于操作者和环境下进行。该发射电离辐射的含同位素化合物可能是有害的在制度和国家层面控制它们的使用对人类健康和严格的许可证制度和法规。在此协议中的实验进行了以下培训开源辐射利用在天主教鲁汶大学（KU鲁汶），并...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+Kinase+Activity+Is+Dependent+on+LRRK2+GTP+Binding+Capacity+but+Independent+of+LRRK2+GTP+Binding.">LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding.</a> PLoS One. 6, 23207-23 (2011).</li><li>Dani&#235;ls, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+mode+of+action+of+the+LRRK2+Y1699C+pathogenic+mutant.">Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant.</a> J Neurochem. 116, 304-315 (2011).</li><li>Civiero, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+highly+purified+leucine-rich+repeat+kinases+1+and+2+demonstrates+formation+of+homodimers.">Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.</a> PLoS One. 7, e43472 (2012).</li><li>Taymans, J. 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B. <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+LRRK2+link+enhanced+GTP-binding+and+kinase+activities+to+neuronal+toxicity.">Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity.</a> Hum.Mol.Genet. 16, 223-232 (2007).</li><li>Sheng, Z., 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=Ser1292+autophosphorylation+is+an+indicator+of+LRRK2+kinase+activity+and+contributes+to+the+cellular+effects+of+PD+mutations.">Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.</a> Sci Transl Med. 4, 164ra161 (2012).</li><li>Li, X., 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=Phosphorylation-dependent+14-3-3+binding+to+LRRK2+is+impaired+by+common+mutations+of+familial+Parkinson's+disease.">Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease.</a> PLoS One. 6, e17153 (2011).</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 (910), 405-413 (2010).</li></ol>

本文提供了一个基本协议，使用代谢标记与32 P-正磷酸盐测定LRRK1和LRRK2的总体磷酸化水平的细胞系。总体战略是直截了当的。亲和标签的LRRK蛋白被表达在HEK293T细胞暴露于含32 P-正磷酸盐介质。的32 P-正磷酸盐的细胞只有几个小时的潜伏期和在含磷酸盐的小区中的所有分子之后被吸收，从而被放射性标记。亲和标签（3XFLAG），然后用于通过免疫沉淀来分离从其它细胞组分的L...

富含亮氨酸重复激酶1和2（LRRK1和LRRK2）是多域的旁系同源共享一个类似的结构域组织。这两个蛋白编码GTP酶的序列类似的Ras家族GTP酶（的Ras复合物的蛋白质，或者ROC）的以及ROC域（COR）的C-末端，从而有效地划分这两种蛋白的ROCO蛋白家族5,6。中华民国-COR域串联的N端，这两个蛋白编码一个富含亮氨酸重 ​​复结构域以及一个锚蛋白样结构域，而只有LRRK2基因编码一个额外的犰狳domein 6-8。 ROC-COR的C-末端，这两个蛋白共享一个丝氨酸-苏氨酸激酶结构域，而只有LRRK2编码的C-末端区8一WD40结构域。 LRRK1和LRRK2的确切蜂窝作用还没有被阐明，但LRRK1有牵连的酪氨酸激酶受体信号1,2，而遗传证据表明，对LRRK2在帕金森病3,4的发病机制中的作用。 <p类=“jove_content”&gt;蛋白质的磷酸化是细胞中一种常见的监管机制。例如，磷酸化可能是必需的酶的活化或蛋白质的募集到信令复杂。 LRRK2的细胞磷酸化已被广泛表征和phosphosite映射已经表明大多数的细胞磷酸化位点发生在锚蛋白重复和富亮氨酸重 ​​复结构域9-11之间的集群。虽然LRRK1细胞磷酸化位点还没有被映射，利用免疫沉淀LRRK1蛋白质印迹的磷蛋白染色从COS7细胞的研究的证据表明，LRRK1蛋白被磷酸化的细胞12。 本文提供了一个基本协议，使用代谢标记与32 P-正磷酸盐测定LRRK1和LRRK2的总体磷酸化水平的细胞系。总体战略是直截了当的。亲和标记LRRK保护制服插件均以HEK293T细胞，这些细胞暴露于含32 P-正磷酸盐媒体。的32 P-正磷酸盐的细胞只有几个小时的潜伏期和在含磷酸盐的小区中的所有分子之后被吸收，从而被放射性标记。亲和标签（3XFLAG），然后用于通过免疫沉淀来分离从其它细胞组分的LRRK蛋白质。免疫沉淀，然后通过SDS-PAGE分离，印迹到PVDF膜上，并掺入的磷酸盐的分析是通过放射自显影对印迹的蛋白质（32 p信号）和western检测（蛋白信号）进行的。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phosphorus-32 Radionuclide, 1 mCi, buffer disodiumphosphate in 1 ml water</td>
 <td>Perkin Elmer</td>
 <td>NEX011001MC</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose)</td>
 <td>Invitrogen</td>
 <td>11971-025</td>
 <td>This medium contains no phosphates</td>
 </tr>
 <tr>
 <td>Anti Flag M2 affiinty gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td>For an equivalent product with red colored gel (useful to more easily visualize the beads), use cat. No. F2426.</td>
 </tr>
 <tr>
 <td>Extra thick blotting filter</td>
 <td>Bio-Rad</td>
 <td>1703965</td>
 <td></td>
 </tr>
 <tr>
 <td>Ponceau S solution</td>
 <td>Sigma</td>
 <td>P7170</td>
 <td></td>
 </tr>
 </tbody>
</table>

metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate

富含亮氨酸的重复激酶1和2（LRRK1和LRRK2）是旁系同源物共享一个类似的结构域组织，包括丝氨酸 - 苏氨酸激酶结构域，复杂的蛋白质结构域的RAS（ROC），ROC域（COR）的C-末端，和富含亮氨酸和锚蛋白样重复的N-末端。 LRRK1和LRRK2的确切蜂窝作用还没有被阐明，但LRRK1有牵连的酪氨酸激酶受体信号1,2，而LRRK2被牵连在帕金森病3,4的发病机制。在这份报告中，我们提出了一个协议来标记LRRK1和LRRK2的蛋白质在细胞中与32磷正磷酸盐，从而提供测量这些2蛋白在细胞中的整体磷酸化水平的一种手段。简言之，亲和标签的LRRK蛋白被表达在HEK293T细胞暴露于含32 P-正磷酸盐介质。的32 P-正磷酸盐的细胞之后被吸收只有几孵化小时，在含有磷酸盐在细胞的所有分子，从而放射性标记。通过亲和标签（3XFLAG）的LRRK蛋白通过免疫沉淀从其它细胞组分中分离。免疫沉淀，然后通过SDS-PAGE分离，印迹到PVDF膜上，并掺入的磷酸盐的分析是通过放射自显影对印迹的蛋白质（32 p信号）和western检测（蛋白信号）进行的。该协议可以很容易地适于监测磷酸化，可以在细胞中表达和分离免疫沉淀的任何其他蛋白质。

<html>为了在细胞中比较LRRK1和LRRK2的总体磷酸化水平，3XFLAG标签LRRK1和LRRK2的表达在HEK293T细胞15。细胞培养在6孔板中，并用32 P标记和上述协议中的文本所描述的分析， 图1示出了代表性的结果为LRRK1和LRRK2的HEK293T细胞代谢标记。放射性磷酸并入观察到两个LRRK1和LRRK2。经32 P水平归一化到如通过用抗flag抗体免疫检测的光密度分析测定的蛋白质水平的定量分析，结果...

Watch this Scientific Journal Video about Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate at JoVE.com

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

富含亮氨酸的重复激酶1和2（LRRK1和LRRK2）是多结构域蛋白，其编码GTP酶都和激酶结构域和它们磷酸化的细胞中。在这里，我们提出了一个协议，以32磷正磷酸盐标记LRRK1和LRRK2的细胞，从而提供衡量其整体的细胞磷酸化水平的一种手段。

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

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

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