Department of Vertebrate Genomics, Max-Planck Institute for Molecular Genetics, Berlin, Germany.
BMC Cancer. 2012 Jan 25;12:38. doi: 10.1186/1471-2407-12-38.
The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level.
We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure.
We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain.
We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.
热休克蛋白 90（Hsp90）是许多信号激酶稳定性所必需的。作为癌症治疗的靶点，它允许同时抑制几条信号通路。然而，其在健康细胞中的抑制也可能导致严重的副作用。这是首次在蛋白质组水平上对 Hsp90 抑制的反应进行全面分析。
我们通过基于固定的广谱激酶抑制剂（“激酶珠”）的亲和蛋白质组学，定量分析了geldanamycin 对一个原代（Hs68）和三个肿瘤细胞系（SW480、U2OS、A549）的激酶组的抑制作用。为了鉴定受影响的途径，我们使用了 KEGG（京都基因与基因组百科全书）途径分类。我们将 Hsp90 和蛋白酶体抑制作用结合起来，鉴定了 Hs68 和 SW480 细胞中的 Hsp90 底物。使用下一代测序确定了来自使用的细胞系的激酶的突变状态。Hsp90 候选客户 RIPK2 的突变被映射到其结构上。
我们测量了 Geldanamycin 处理后来自四个细胞系的 >140 种蛋白激酶的相对丰度，并鉴定了许多新的潜在 Hsp90 底物。这些激酶代表了不同的家族和细胞功能，其中强烈代表了肿瘤进展相关的途径，如 BMP、MAPK 和 TGF-β 信号级联。与蛋白酶体抑制剂 MG132 共同处理使我们能够将 64 种激酶分类为真正的 Hsp90 客户。最后，7 种激酶的突变与对 Hsp90 抑制的反应改变相关。候选客户 RIPK2 的结构建模表明突变对提议的 Hsp90 结合域有影响。
我们提出了一个高可信度的 Hsp90 激酶客户列表，这为靶向和组合癌症治疗以及诊断应用提供了新的机会。
