Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates
Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates
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DOI：
10.1371/journal.pone.0052747
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发表时间：
2012-12-26
期刊：
影响因子：
3.7
通讯作者：
Schwartz, Daniel
中科院分区：
文献类型：
--
作者：
Chou, Michael F.;Prisic, Sladjana;Schwartz, Daniel
蛋白激酶靶点的确定仍然是我们理解正常和病变细胞状态下信号转导的一个重要瓶颈。激酶通过底物蛋白上的序列基序部分地识别它们的底物，迄今为止，已经使用组合肽库方法最有效地阐明了底物蛋白上的序列基序。在这里，我们提出并证明了ProPeL的方法，通过使用天然细菌蛋白质组，作为体内库的数千个同时磷酸化反应的激酶特异性基序的简单和准确的发现。利用在E.大肠杆菌，然后通过质谱分析，该方法准确地概括了人类嗜碱性（蛋白激酶A）和嗜酸性（酪蛋白激酶II）激酶的良好建立的基序偏好。这些基序，来自PKA和CK II使用细菌序列数据，然后进一步验证，利用它们与scan-x软件程序结合计算预测已知的人类磷酸化位点的高置信度。
The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.