Design and use of analog-sensitive protein kinases.
Design and use of analog-sensitive protein kinases.
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DOI：
10.1002/0471142727.mb1811s66
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发表时间：
2004-05-01
影响因子：
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通讯作者：
Weiss, Eric L
中科院分区：
文献类型：
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作者：
Blethrow, Justin;Zhang, Chao;Weiss, Eric L
许多蛋白激酶可以被设计为接受野生型激酶不能有效利用的 ATP 类似物。这些工程化激酶被称为“类似物敏感”或“-as”等位基因，通常也对不阻断非突变激酶活性的蛋白激酶抑制剂变体敏感。体外选择性使用α-as激酶的放射性标记的ATP类似物可用于鉴定复杂提取物中单个激酶的直接磷酸化靶标。在用工程化等位基因取代野生型激酶基因是可行的生物体中，α-as激酶的体内活性可以用等位基因特异性抑制剂可逆地阻断。因此，类似物敏感激酶可以成为发现单个酶的细胞功能和磷酸化靶点的有效工具。讨论了这些等位基因的设计和使用的理论背景，以及构建任何激酶的候选等位基因的策略。
Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild-type kinases. These engineered kinases, which are referred to as "analog-sensitive" or "-as" alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by -as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild-type kinase genes with engineered alleles, the in vivo activity of a -as kinase can be reversibly blocked with an allele-specific inhibitor. Thus, analog-sensitive kinases can be effective tools for discovery of the cellular functions and phosphorylation targets of individual enzymes. A theoretical background for the design and use of these alleles is discussed, as are strategies for construction of candidate -as alleles of any kinase.