A combinatorial approach to identifying protein tyrosine phosphatase substrates from a phosphotyrosine peptide library
A combinatorial approach to identifying protein tyrosine phosphatase substrates from a phosphotyrosine peptide library
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DOI：
10.1021/ja971825k
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发表时间：
1997-10-08
影响因子：
15
通讯作者：
Balasubramanian, S
中科院分区：
文献类型：
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作者：
Cheung, YW;Abell, C;Balasubramanian, S
蛋白质磷酸化在细胞功能如生长和分化的调节中起着关键作用。1蛋白激酶和蛋白磷酸酶的底物识别研究对我们理解蛋白磷酸化至关重要，并且可以产生有价值的结构信息，以帮助设计干扰细胞信号传导过程的选择性抑制剂。蛋白酪氨酸磷酸酶（PTPs）使用磷酸酪氨酸肽底物的研究已经证明了一定程度的底物特异性。2，3这些研究主要是基于肽序列来源于磷蛋白，包括自磷酸化位点的胰岛素受体4，5和表皮生长因子受体。2，3，6然而，合成和测试单个磷酸肽的必要性限制了PTP特异性的探索。合适的组合方法将提供快速鉴定PTP的底物共有序列的有价值的手段。有应用液相组合方法发现与靶受体结合的配体的实例。7对于固相文库，配体选择已经通过使用荧光标记的靶蛋白8的结合测定或通过对蛋白结合珠进行免疫染色来介导。9-11识别良好的底物酶周转需要区分固相催化产物的固相底物，单珠灵敏度。这已经在通过淬灭荧光测定12筛选具有内肽酶的肽库以及通过掺入放射性32P检测蛋白激酶底物中得到证实。13固相产物的免疫检测已用于催化抗体的选择。14我们报告了一种新的组合方法，用于从磷酸酪氨酰肽的树脂结合文库中鉴定PTP的底物。
Protein phosphorylation plays a pivotal role in the regulation of cellular functions such as growth and differentiation. 1 The study of the substrate recognition of protein kinases and protein phosphatases is critical to our understanding of protein phosphorylation and could yield valuable structural information to assist in the design of selective inhibitors to interfere with cellular signalling processes. Studies on protein tyrosine phosphatases (PTPs) using phosphotyrosyl peptide substrates have demonstrated some level of substrate specificity. 2, 3 Such studies have largely been based on peptide sequences derived from phosphoproteins that include the autophosphorylation sites of the insulin receptor4, 5 and the epidermal growth factor receptor. 2, 3, 6 However, the necessity to synthesize and test single phosphopeptides limits the exploration of PTP specificities. A suitable combinatorial approach would provide a valuable means of rapidly identifying substrate consensus sequences for PTPs. There are examples of the application of solution-phase combinatorial methods to the discovery of ligands that bind to a target receptor. 7 For a solid-phase library, ligand selection has been mediated by binding assays using a fluorescently labeled target protein8 or by immunostaining the protein-bound beads. 9-11 The identification of good substrates for enzymatic turnover requires the discrimination of solid-phase products of catalysis from solid-phase substrates, with single-bead sensitivity. This has been demonstrated in the screening of peptide libraries with endopeptidases by a quenched fluorescence assay12 and also in the detection of protein kinase substrates by the incorporation of radioactive 32P. 13 Immunodetection of solidphase products has been employed in the selection of catalytic antibodies. 14 We report a novel combinatorial approach for identifying substrates for PTPs from a resin-bound library of phosphotyrosyl peptides.