O'Neill T, Dwyer A J, Ziv Y, Chan D W, Lees-Miller S P, Abraham R H, Lai J H, Hill D, Shiloh Y, Cantley L C, Rathbun G A
Center for Blood Research, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2000 Jul 28;275(30):22719-27. doi: 10.1074/jbc.M001002200.
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.
共济失调毛细血管扩张症突变（ATM）基因编码一种丝氨酸/苏氨酸蛋白激酶，该激酶在基因组监测和发育中起关键作用。在此，我们使用肽库方法来确定ATM激酶活性的体外底物特异性。肽库分析确定了一个最佳序列，其核心基序为LSQE，该序列优先被ATM磷酸化。通过使用特定的肽库或单个肽底物来评估LSQE基序中丝氨酸周围氨基酸的作用。构成LSQE序列的所有氨基酸对于ATM的最大肽底物适用性都至关重要。将DNA依赖性蛋白激酶（DNA-PK），一种与ATM相关且在DNA修复中起重要作用的丝氨酸/苏氨酸激酶，在肽底物选择性方面与ATM进行了比较。发现DNA-PK在对磷酸化丝氨酸相邻氨基酸的偏好方面具有独特性。肽库分析确定了ATM的一个优选氨基酸基序，该基序允许在ATM和DNA-PK激酶活性之间进行明确区分。使用源自库的ATM序列进行数据库搜索，鉴定出了先前已表征的ATM底物以及可能在ATM指导的信号通路下游起作用的新型候选底物靶点。
