A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870
A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870
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DOI：
10.1042/bsr20130094
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发表时间：
2014-01-01
影响因子：
4
通讯作者：
Zaru, Rossana
中科院分区：
文献类型：
--
作者：
Edgar, Alexander J.;Trost, Matthias;Zaru, Rossana
蛋白激酶抑制剂通常具有不能完全归因于预期靶激酶的有趣作用，但鉴定可能解释该作用的其他靶点并不简单。通过比较Rsk（p90核糖体S6激酶）激酶的两种不同抑制剂，我们发现越来越多地使用的化合物BI-D1870在小鼠DC（树突状细胞）中具有生物学效应，不能单独归因于Rsk或其他记录的靶点。我们评估了BI-D1870和第二种Rsk抑制剂BIX 02565保护酶活性位点不与生物素化核苷酸酰基磷酸反应的能力。使用SILAC（细胞培养物中氨基酸的稳定同位素标记）标记的DC裂解物作为酶靶点的来源，我们鉴定了几种与BI-D1870相互作用但不与BIX 02565相互作用的激酶。我们证实，这些激酶，包括Slk，Lok和Mst 1，被BI-D1870抑制，但BIX 02565的抑制程度要小得多，并且在活细胞中，BI-D1870阻断了它们的一些底物的磷酸化。我们的研究结果表明，BI-D1870抑制剂应谨慎使用。我们使用的SILAC为基础的方法应该是有用的，进一步比较无偏的分析与有趣的生物学效应的激酶抑制剂的目标频谱的条件下，密切模仿细胞中发现的。
Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells.