Global peptide microarray profiling of tyrosine kinases deregulated in cancer

描述(申请人提供)：蛋白酪氨酸激酶(PTK)在人类癌症中发挥关键作用，是一大类新兴抗癌药物的靶标。在单个肿瘤中，多个PTK是活跃的，并且大量的PTK对于维持转化的表型是必不可少的。虽然通过质谱学(MS)已经在癌细胞中定位了大量的磷酸化位点，但使这些位点磷酸化的特定激酶的身份仍是未知的，只有极少数例外。我们建议使用新兴的多肽微阵列技术来鉴定整个人类PTK的共同磷酸化序列。我们将构建一组哺乳动物表达载体，产生与谷胱甘肽S转移酶融合的每个PTK。每个PTK将从哺乳动物细胞过度表达系统中进行亲和纯化，并进行多肽芯片筛选。这些筛选将揭示每一种激酶在其目标底物中的磷酸化位置所偏好的特定序列。我们将使用这些数据从癌细胞中挖掘磷酸蛋白质组数据，将已知的磷酸化位点与它们各自的激酶联系起来。预测的激酶-底物关系将通过siRNA敲除癌细胞中的相关激酶而得到验证。这项工作的广泛目标是阐明癌细胞中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理解，提供对激酶靶向治疗作用机制的洞察，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.