Zhang Chao, Kenski Denise M, Paulson Jennifer L, Bonshtien Arale, Sessa Guido, Cross Janet V, Templeton Dennis J, Shokat Kevan M
Department of Cellular and Molecular Pharmacology, University of California San Francisco, 600 16th Street, San Francisco, California 94143-2280, USA.
Nat Methods. 2005 Jun;2(6):435-41. doi: 10.1038/nmeth764.
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 nucleotide 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-terminal 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.
蛋白激酶的化学遗传学分析涉及对激酶进行工程改造，使其对野生型激酶无法识别的抑制剂和ATP类似物具有独特的敏感性。尽管这种方法已成功应用于二十多种激酶，但仍有几种激酶无法耐受对ATP结合口袋进行的必要修饰，因为在控制抑制剂和核苷酸底物特异性的“守门人”残基发生突变后，它们会失去催化活性或细胞功能。在此，我们描述了通过鉴定第二位点抑制突变来挽救“不耐受”激酶活性的方法。利用氨基糖苷激酶APH(3')-IIIa进行细菌遗传筛选以寻找第二位点抑制子，结果在激酶结构域中发现了几个抑制热点。基于该筛选结果，我们将重点放在N端亚结构域的β折叠上，并生成了该区域蛋白激酶基于结构的序列比对。通过该比对，我们为包括Cdc5、MEKK1、GRK2和Pto在内的几种不同激酶鉴定出了第二位点抑制子。鉴定第二位点抑制子以挽救不耐受激酶活性的能力，应有助于对基因组中大多数蛋白激酶进行化学遗传学分析。
