A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases
A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases
复制标题
DOI：
10.1038/nmeth764
复制
发表时间：
2005-06-01
期刊：
影响因子：
48
通讯作者：
Shokat, KM
中科院分区：
文献类型：
--
作者：
Zhang, C;Kenski, DM;Shokat, KM
蛋白激酶的化学遗传分析涉及对抑制剂和ATP类似物具有独特敏感性的工程激酶，这些抑制剂和ATP类似物不被野生型激酶识别。尽管这种方法成功地应用于二十多种激酶，但有几种激酶不能耐受对ATP结合袋进行必要的修饰，因为当控制抑制剂和核苷酸底物特异性的“看门人”残基突变时，它们会失去催化活性或细胞功能。在这里，我们描述了第二位点抑制突变的鉴定，以挽救“不耐受”激酶的活性。利用氨基糖苷激酶APH（3’）-IIIa对第二位点抑制因子的细菌遗传选择揭示了激酶结构域的几个抑制热点。根据这一选择的结果，我们将重点放在n端亚域的β片上，并在该区域生成了基于结构的蛋白激酶序列比对。从这个比对中，我们确定了几种不同激酶的第二位点抑制因子，包括Cdc5、MEKK1、GRK2和Pto。鉴定第二位点抑制因子以挽救不耐受激酶活性的能力将有助于基因组中大多数蛋白激酶的化学遗传分析。
Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucteotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminai subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.