Peptide reporters of kinase activity in whole cell lysates.
Peptide reporters of kinase activity in whole cell lysates.
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DOI：
10.1002/bip.21401
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Kron, Stephen J.
中科院分区：
文献类型：
--
作者：
Wu, Ding;Sylvester, Juliesta E.;Parker, Laurie L.;Zhou, Guangchang;Kron, Stephen J.
激酶测定用于筛选小分子抑制剂，这些抑制剂可能显示出作为靶向药物治疗的前景。使用细胞裂解物而不是纯化的激酶提供了在生物环境中抑制剂敏感性和选择性的更准确的估计。这篇综述总结了一系列的均相（溶液相）和异质性（固体支持）的格式可用于使用肽底物来监测细胞裂解物中的激酶活性。重点是异质性激酶测定，肽底物肽被用作模型来优化呈现几何形状和用于激酶识别的短序列的模块化排列。我们目前的结果固定在二维和三维表面，如96孔板和载玻片上的水凝胶，荧光Luminex珠的肽。我们讨论的方法，以提高检测灵敏度，使用化学荧光ELISA，抗体为基础的识别，和无标记质谱。监测细胞裂解物中特定激酶的活性提出了挑战，可以通过操纵肽底物以优化测定条件来克服这些挑战。特别地，通过1）在基底和表面之间添加长支链亲水性接头，2）改变肽相对于表面的取向，和3）包括顺式或反式的肽配体以将激酶募集到表面来改善信号与背景比。通过提高固定化肽底物在溶液中的激酶的可及性，磷酸化的表观速率增加，测定对内源性激酶活性的变化更敏感。这些策略可以推广到改善大多数肽底物的反应性，用于异源激酶测定与细胞裂解物。
Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.
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DOI：
10.1016/j.bbapap.2003.11.012
发表时间：
2004-03-11
影响因子：
3.2
作者：
Chen, CA;Yell, RH;Lawrence, DS
通讯作者：
Lawrence, DS
DOI：
10.1073/pnas.89.6.2365
发表时间：
1992-03-15
影响因子：
11.1
作者：
HAYCOCK, JW;AHN, NG;KREBS, EG
通讯作者：
KREBS, EG
影响因子：
2.9
作者：
Bozinovski, S;Cristiano, BE;Pearson, RB
通讯作者：
Pearson, RB
影响因子：
--
作者：
Han, Xiaoming;Yamanouchi, Go;Katayama, Yoshiki
通讯作者：
Katayama, Yoshiki
影响因子：
16.6
作者：
Johnson, Erik C. B.;Durek, Thomas;Kent, Stephen B. N.
通讯作者：
Kent, Stephen B. N.