Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE
Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE
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DOI：
10.1002/pmic.200800243
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发表时间：
2008-08-01
期刊：
影响因子：
3.4
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Kinoshita, Eiji;Kinoshita-Kikuta, Emiko;Koike, Tohru
在此，我们证明了分离的磷蛋白同种型具有相同数量的磷酸基团，使用磷酸盐亲和SDS-PAGE。磷酸盐亲和位点是聚丙烯酰胺结合的Phos标签，其使得能够从其非磷酸化对应物中检测磷蛋白的迁移率变化。作为分离的第一个实际例子，我们通过三种酪氨酸激酶c-Abl、MET和Fyn中的每一种来表征单磷酸化Tau同种型。在Tyr-394、Tyr-197或Tyr-18处磷酸化的每个单磷酸同种型被检测为三个不同的迁移条带。作为进一步的应用，我们将该技术扩展到苜蓿中华根瘤菌FixL/FixJ双组分系统中His和Asp磷酸化同种型的迁移率变化分析。FixL在His-285处被ATP自磷酸化，磷酸基团被转移到FixJ的Asp-54，随后被FixL磷酸酶活性去除。使用这种方法，我们首先进行了同时检测的磷酸化和非磷酸化的同种型的FixL和FixJ在体外磷酸转移反应中产生的。结果，确认了含有磷酸化His残基的FixL的单磷酸同种型。另一方面，对于FixJ，检测到两个单磷酸化同种型作为两个不同的迁移带。一种是众所周知的在Asp-54磷酸化的同种型。另一种是在His-84处磷酸化的新型同种型。
Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr-394, Tyr-197, or Tyr-18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two-component system. FixL is autophosphorylated at the His-285 with ATP, and the phosphate group is transferred to the Asp-54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well-known isotype phosphorylated at the Asp-54. The other is a novel isotype phosphorylated at the His-84.