Identification of peptidic substrates for the human kinase Myt1 using peptide microarrays.
Identification of peptidic substrates for the human kinase Myt1 using peptide microarrays.
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DOI：
10.1016/j.bmc.2015.05.021
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发表时间：
2015-08
影响因子：
3.5
通讯作者：
A. Rohe;Charlott Platzer;A. Masch;Sandra Greiner;Claudia Henze;C. Ihling;F. Erdmann;M. Schutkowski;W. Sippl;Matthias Schmidt
中科院分区：
文献类型：
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作者：
A. Rohe;Charlott Platzer;A. Masch;Sandra Greiner;Claudia Henze;C. Ihling;F. Erdmann;M. Schutkowski;W. Sippl;Matthias Schmidt
Myt1是Wee-Kinase家族的一员，参与细胞周期的G2/M检查点调控。到目前为止，还没有报道适合于基于活性的筛选的多肽底物，这阻碍了Myt1激酶抑制剂的系统开发。Myt1抑制剂必须通过结合试验或与昂贵的蛋白质底物的活性试验来鉴定。在这里，多肽微阵列方法被用来鉴定多肽Myt1底物。使用相同的技术对Wee1激酶进行了比对。多肽芯片实验中的Myt1点击通过荧光偏振分析在溶液中得到验证，并鉴定了几种来源于细胞蛋白的多肽底物。随后，通过MS裂解研究进行了磷酸化位点的确定，并通过激酶抑制剂谱对鉴定的底物进行了验证。
Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.