A chemical switch for inhibitor-sensitive alleles of any protein kinase
A chemical switch for inhibitor-sensitive alleles of any protein kinase
复制标题
DOI：
10.1038/35030148
复制
发表时间：
2000-09-21
期刊：
影响因子：
64.8
通讯作者：
Shokat, KM
中科院分区：
文献类型：
--
作者：
Bishop, AC;Ubersax, JA;Shokat, KM
蛋白激酶已被证明在很大程度上抵抗高度特异性抑制剂的设计，即使在组合化学的帮助下（1）。这些试剂的缺乏使为单个激酶分配特定信号作用的努力变得复杂。在这里，我们描述了一种化学遗传策略，使蛋白激酶对不抑制野生型激酶的细胞渗透性分子敏感（2）。从两个抑制剂支架，我们已经确定了有效的和选择性的抑制剂，从五个不同的亚家族致敏激酶。酪氨酸和丝氨酸/苏氨酸激酶同样适用于这种方法。我们已经分析了一个芽殖酵母菌株携带的细胞周期蛋白依赖性激酶Cdc 28（CDK 1）的地方的野生型蛋白质的一个通道敏感的形式。体内Cdc 28的特异性抑制导致有丝分裂前细胞周期停滞，这与通常在温度敏感性cdc 28突变体中观察到的G1停滞不同（3）。赋予寡核苷酸敏感性的突变很容易从一级序列比对中识别。因此，这种方法可用于系统地产生蛋白激酶的条件等位基因，允许快速功能表征这个重要的基因家族的成员。
Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry(1). The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wildtype kinases(2). From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants(3). The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.