Peptide and protein library screening defines optimal substrate motifs for AKT/PKB
Peptide and protein library screening defines optimal substrate motifs for AKT/PKB
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DOI：
10.1074/jbc.m005497200
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发表时间：
2000-11-17
影响因子：
4.8
通讯作者：
Cantley, LC
中科院分区：
文献类型：
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作者：
Obata, T;Yaffe, MB;Cantley, LC
AKT最初被鉴定为具有Pleckstrin同源性和Ser/Thr蛋白激酶结构域的原癌基因。最近的研究表明，AKT调节多种细胞功能，包括细胞生存、细胞生长、细胞分化、细胞周期进程、转录、翻译和细胞代谢。为了阐明AKT的底物特异性，我们使用定向肽库方法确定了N-端和C-端到磷酸化位置的最佳氨基酸。预测的最佳多肽底物(Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly，其中Ser*是磷酸化位点)与我们先前为相关嗜碱性蛋白激酶定义的最佳底物相似但不同：如蛋白激酶A、富Ser/Arg的蛋白激酶和蛋白激酶C家族成员。V-max/K-m比值最高的部位是Arg-3和gt；Arg-5和gt；Arg-7。通过筛选Lambda Gex噬菌体HeLa细胞表达文库，进一步研究了AKT的底物特异性。该方法鉴定的所有底物均含有Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)基序，与多肽文库筛选鉴定的基序一致。这项研究的结果应该有助于从初级序列中预测可能的AKT底物。
AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify:the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such: as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V-max/K-m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambda GEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from Primary sequences.