Global peptide microarray profiling of tyrosine kinases deregulated in cancer

描述（由申请人提供）：蛋白酪氨酸激酶（PTKs）在人类癌症中起着关键作用，是一类主要的新兴抗癌药物的靶点。在单个肿瘤中，多个ptk是活跃的，并且相当数量的ptk对于维持转化表型是必不可少的。虽然通过质谱（MS）已经在癌细胞中绘制了大量的磷酸化位点，但磷酸化这些位点的特定激酶的身份却很少有例外。我们建议使用新兴的肽微阵列技术来鉴定整个人类PTKs的一致磷酸化序列。我们将生成一组哺乳动物表达载体，产生融合谷胱甘肽s -转移酶的每一个PTK。每个PTK将从哺乳动物细胞过表达系统亲和纯化，并进行肽芯片筛选。这些筛选将揭示每个激酶在其靶底物磷酸化位点的特定序列。我们将利用这些数据挖掘来自癌细胞的磷酸化蛋白质组学数据，将已知的磷酸化位点与其各自的激酶联系起来。预测的激酶-底物关系将通过在癌细胞系中敲低相关激酶的siRNA来验证。这项工作的主要目标是阐明癌细胞磷酸化网络中的关键连接。这些研究将丰富我们对细胞转化和肿瘤维持的基本机制的认识，为激酶靶向治疗的作用机制提供新的见解，并为治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Protein tyrosine kinases (PTKs) play pivotal roles in human cancer and are the targets of a major class of emerging anti-cancer drugs. In a single tumor, multiple PTKs are active and a substantial number are essential for maintaining the transformed phenotype. Though large numbers of phosphorylation sites have been mapped in cancer cells through mass spectrometry (MS), the identity of the specific kinases that phosphorylate these sites are with very few exceptions unknown. We propose to use emerging peptide microarray technology to identify consensus phosphorylation sequences for the entire set of human PTKs. We will generate a set of mammalian expression vectors producing every PTK fused to glutathione S-transferase. Each PTK will be affinity purified from a mammalian cell overexpression system and subjected to peptide microarray screening. These screens will reveal specific sequences preferred by each kinase at phosphorylation sites in their target substrates. We will use this data to mine phosphoproteomics data from cancer cells to connect known sites of phosphorylation to their respective kinases. Predicted kinase-substrate relationships will be validated through siRNA knockdown of the relevant kinase in cancer cell lines. The broad goal of this work is to elucidate critical connections in phosphorylation networks in cancer cells. These studies will enrich our understanding of the basic mechanisms of cellular transformation and tumor maintenance, provide insight into the mechanisms of action of kinase-targeted therapeutics, and suggest new targets for therapeutic intervention.