Bonn Stefan, Herrero Saturnino, Breitenlechner Christine B, Erlbruch Andrea, Lehmann Wolf, Engh Richard A, Gassel Michael, Bossemeyer Dirk
Group of Structural Biochemistry, German Cancer Research Center, 69120 Heidelberg.
J Biol Chem. 2006 Aug 25;281(34):24818-30. doi: 10.1074/jbc.M512374200. Epub 2006 May 12.
Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.
控制异常激酶介导的细胞信号传导是癌症治疗的主要策略；像特罗凯和格列卫这样成功的蛋白激酶抑制剂证实了这种方法。然而，抑制剂对靶向激酶的特异性是治疗成功的关键因素。基于同源建模，我们之前在Rho激酶的活性位点鉴定出四个氨基酸，它们可能决定了相对于蛋白激酶A（PKA）观察到的Rho激酶抑制剂特异性（按照PKA编号：T183A、L49I、V123M和E127D），还有第五个氨基酸（Q181K），它在PKA-PKB杂合蛋白中发挥了惊人的作用。我们已将PKA中的这些残基系统地突变为Rho激酶中的对应残基，分别进行以及组合进行。使用四种Rho激酶特异性、一种PKA特异性和一种泛激酶特异性抑制剂，我们测量了突变蛋白的抑制剂结合特性，并确定了各个残基作为特异性决定因素的作用。两种包含T183A和L49I突变组合的复合突变蛋白，与Rho激酶非常相似。动力学结果证实了侧链特性构成选择性主要决定因素的假设。分析的一个意外结果是单个突变由简单因素产生的一致贡献。替代激酶抑制剂复合物的晶体结构为理解这些选择性决定残基提供了详细依据。从这些PKA突变体获得动力学和结构数据的能力，再加上它们类似Rho激酶的选择性谱，使它们对于基于结构的抑制剂设计用作替代激酶很有价值。
