Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine pro tein kinase that plays a critical role in genomic surveillance and developm ent. Here, we use a peptide library approach to define the in vitro substra te specificity of ATM kinase activity. The peptide library analysis identif ied an optimal sequence with a central core motif of LSQE that is preferent ially phosphorylated by ATM. The contributions of the amino acids surroundi ng serine in the LSQE motif were assessed by utilizing specific peptide lib raries or individual peptide substrates. All amino acids comprising the LSQ E 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 sub strate selectivity. DNA-PK was found to be unique in its preference of neig hboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinction s between ATM and DNA-PK kinase activity. Data base searches using the libr ary-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstr eam in ATM-directed signaling pathways.