The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta
The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta
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DOI：
10.1016/j.febslet.2008.04.060
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发表时间：
2008-06-11
期刊：
影响因子：
3.5
通讯作者：
Sawasaki, Tatsuya
中科院分区：
文献类型：
--
作者：
Masaoka, Takashi;Nishi, Mayuko;Sawasaki, Tatsuya
钙/钙调蛋白依赖性蛋白激酶II（CaMKII）在介导钙信号传导中起着至关重要的作用。在这里，我们展示了一种方法，用于筛选底物磷酸化的人CaMKII δ使用小麦无细胞系统。将表达CaMKII delta的无细胞混合物与HeLa提取物和放射性标记的ATP一起孵育。从双向电泳凝胶和质谱分析，发现两个蛋白质。基于体外激酶分析的无细胞显示，CaMKII δ磷酸化真核生物翻译起始因子413和应激诱导磷蛋白1（STIP 1），后者Ser 189。此外，在HeLa细胞中，组成型活性CaMKII δ磷酸化STIP 1，并显着促进STIP 1的核定位，表明通过CaMKII δ的钙信号可能调节STIP的亚细胞定位。这种方法可能是一个有用的工具，蛋白激酶的靶标筛选。
Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 413 and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIPI. This approach may be a useful tool for target screening of protein kinases.