Biosensors of protein kinase action: from in vitro assays to living cells
Biosensors of protein kinase action: from in vitro assays to living cells
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DOI：
10.1016/j.bbapap.2003.11.012
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发表时间：
2004-03-11
影响因子：
3.2
通讯作者：
Lawrence, DS
中科院分区：
文献类型：
--
作者：
Chen, CA;Yell, RH;Lawrence, DS
蛋白激酶及其参与的信号转导途径现在被认为是有吸引力的药物靶点。蛋白激酶家族的抑制剂不仅作为治疗剂具有巨大的前景，而且在信号通路的表征中也具有深远的用途。活细胞中蛋白激酶活性的直接可视化提供了对这些抑制剂在生理环境中的功效和选择性的真正评估。此外，能够在真实的时间内可视化蛋白激酶的活性，这使得能够直接测量响应于细胞外刺激的特定信号传导途径的活化。我们已经开发了两个系列的蛋白激酶C（PKC）的荧光底物，使用的策略，位置直接对进行磷酸化的残基的磷酸化基团。第一系列PKC底物部分基于Tsien及其合作者在20世纪80年代开发的Ca+2指标。在这种情况下，底物的磷酸化产生二价金属离子结合位点。金属离子配位。荧光变化通过类似于Ca+2指示剂所述的机制发生。通过制备和随后筛选荧光标记的PKC肽底物文库，鉴定了第二系列PKC传感器。该先导衍生物显示磷酸化诱导的荧光变化，其允许细胞裂解物和活细胞中的实时PKC活性的可视化。此外，免疫耗竭实验表明，荧光标记的肽是选择性的（如果不是唯一的话）。被常规PKC磷酸化。两种蛋白激酶生物传感器策略都利用了肽可以被修饰以创建结构改变的类似物的文库的容易性。然而，肽的固有合成突变性不仅限于文库构建。例如，通过将具有不同生物物理性质的荧光团附着到适当设计的活性定点肽上，最终可能同时监测多种蛋白激酶。(C)2003 Elsevier B.V.保留所有权利。
Protein kinases, and the signal transduction pathways in which they participate, are now recognized to be medicinally attractive targets of opportunity. Inhibitors of the protein kinase family not only hold great promise as therapeutic agents, but are also of profound utility in the characterization of signaling pathways. The direct visualization of protein kinase activity in living cells provides a genuine assessment of the efficacy and selectivity of these inhibitors in a physiological setting. In addition, the ability to visualize the activity of a protein kinase in real time furnishes a direct measurement of the activation of specific signaling pathways in response to extracellular stimuli. We have developed two series of fluorescent substrates for protein kinase C (PKC) using a strategy that positions the reporter-group directly on the residue undergoing phosphorylation. The first series of PKC substrates is based, in part, on the Ca+2 indicators developed by Tsien and his collaborators during the 1980s. In this case, phosphorylation of the substrate creates a divalent metal ion binding site. Upon metal ion coordination. a fluorescence change transpires via a mechanism analogous to that described for the Ca+2 indicators. The second series of PKC sensors was identified via the preparation and subsequent screen of a library of fluorescently-labeled PKC peptide substrates. The lead derivative displays a phosphorylation-induced fluorescence change that allows the visualization of real-time PKC activity in both cell lysates and living cells. Furthermore, immunodepletion experiments demonstrate that the fluorescently-tagged peptide is selectively, if not exclusively.. phosphorylated by the conventional PKCs. Both of the protein kinase biosensor strategies take advantage of the ease with which peptides can be modified to create libraries of structurally altered analogs. However, the inherent synthetic mutability of peptides is not just limited to library construction. For example, it may ultimately be possible to simultaneously monitor multiple protein kinases by affixing fluorophores with distinct photophysical properties to appropriately designed active site-directed peptides. (C) 2003 Elsevier B.V. All rights reserved.