Utilization of oriented peptide libraries to identify substrate motifs selected by ATM
Utilization of oriented peptide libraries to identify substrate motifs selected by ATM
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DOI：
10.1074/jbc.m001002200
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Rathbun, GA
中科院分区：
文献类型：
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作者：
O'Neill, T;Dwyer, AJ;Rathbun, GA
共济失调毛细血管扩张症突变（ATM）基因编码丝氨酸/苏氨酸蛋白激酶，在基因组监视和发育中起着关键作用。在这里，我们使用肽库的方法来定义ATM激酶活性的体外底物特异性。肽库分析确定了一个最佳的序列，其中心核心基序LSQE优先被ATM磷酸化。通过利用特定的肽库或单个肽底物来评估LSQE基序中丝氨酸周围的氨基酸的贡献。包含LSQE序列的所有氨基酸对于ATM的最大肽底物适用性至关重要。DNA依赖性蛋白激酶（DNA-PK）是一种与ATM相关的丝氨酸/苏氨酸激酶，在DNA修复中起重要作用。DNA-PK被发现是独特的，在其邻近的氨基酸的磷酸化丝氨酸的偏好。肽库分析定义了ATM的优选氨基酸基序，其允许ATM和DNA-PK激酶活性之间的明确区别。数据库搜索使用库衍生的ATM序列识别先前表征的ATM底物，以及可能在ATM定向信号通路下游起作用的新的候选底物靶。
The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.