Protein kinase structure and function analysis with chemical tools
Protein kinase structure and function analysis with chemical tools
复制标题
DOI：
10.1016/j.bbapap.2005.08.020
复制
发表时间：
2005-12-30
影响因子：
3.2
通讯作者：
Cole, PA
中科院分区：
文献类型：
--
作者：
Shen, K;Hines, AC;Cole, PA
蛋白激酶是参与细胞信号转导的最大的酶超家族，并且代表了一系列疾病的治疗靶标。许多实验室一直在努力了解它们的催化机制，发现抑制剂并识别它们的细胞功能。在这篇综述中，我们将描述两种用于分析蛋白激酶的方法：双底物类似物抑制和膦酸酯类似物利用。这两种方法都与蛋白质半合成方法表达的蛋白质连接结合使用，以促进我们对激酶-底物相互作用和磷酸化功能阐明的理解。先前关于蛋白激酶机制的性质的工作表明它遵循解离过渡态。针对胰岛素受体激酶设计了一种双底物类似物，以模拟解离过渡态反应坐标距离的几何形状。这种双底物化合物被证明是一种有效的抑制剂对胰岛素受体激酶和占领肽和核苷酸结合位点。双底物化合物与改变氢键的潜力，以及不同的间隔之间的腺嘌呤和肽证明了原始设计功能的重要性。我们还表明，相关的双底物类似物可用于有效地阻断丝氨酸/苏氨酸激酶，包括蛋白激酶A。由于许多蛋白激酶识别折叠的蛋白质底物以进行有效磷酸化，因此将肽-ATP缀合物掺入蛋白质结构中是有利的。使用表达的蛋白质连接，产生Src-ATP缀合物，并显示为Csk酪氨酸激酶的高亲和力配体。磷酸丝氨酸/磷酸酪氨酸的非水解模拟物可用于检查磷酸化事件的功能。使用表达的蛋白质连接，我们已经使用膦酰基亚甲基苯丙氨酸和膦酰基亚甲基丙氨酸分别探测Tyr和Ser的磷酸化。这些工具允许分析的SH 2-磷酸酶（SHP 1和SHP 2），揭示了一种新的分子内刺激的催化活性介导的相应的磷酸化事件。它们也被用于表征通过磷酸化对褪黑激素节律酶的细胞调节。(c)2005 Elsevier B. V.保留所有权利。
Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase- substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation. (c) 2005 Elsevier B.V. All rights reserved.