Global peptide microarray profiling of tyrosine kinases deregulated in cancer

癌症中酪氨酸激酶失调的整体肽微阵列分析

• 批准号: 8054196
• 负责人: BENJAMIN E TURK
• 金额: $ 16.95万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054196
• 关键词: Affinity; Antineoplastic Agents; Biological Assay; Cancer cell line; Chimeric Proteins; Consensus; Data; Data Set; Detection; Glass; Glutathione S-Transferase; Goals; Human; In Vitro; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Methods; Microarray Analysis; Mining; Non-Small-Cell Lung Carcinoma; Peptide Library; Peptides; Phenotype; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein Tyrosine Kinase; Protocols documentation; Research; Role; Screening procedure; Site; Slide; Small Interfering RNA; System; Testing; Therapeutic Intervention; Tyrosine Phosphorylation Site; Work; cancer cell; cancer therapy; density; expression vector; insight; metaplastic cell transformation; miniaturize; overexpression; therapeutic target; tumor

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.

描述（由申请人提供）：蛋白酪氨酸激酶（PTK）在人类癌症中起关键作用，是一类主要的新兴抗癌药物的靶点。在单个肿瘤中，多种PTK是活性的，并且大量PTK对于维持转化的表型是必需的。虽然大量的磷酸化位点已经通过质谱（MS）在癌细胞中定位，但磷酸化这些位点的特定激酶的身份是未知的，只有极少数例外。我们建议使用新兴的肽微阵列技术，以确定整个人类PTKs的共识磷酸化序列。我们将生成一组哺乳动物表达载体，其产生与谷胱甘肽S-转移酶融合的每个PTK。每种PTK将从哺乳动物细胞过表达系统中亲和纯化，并进行肽微阵列筛选。这些筛选将揭示每种激酶在其靶底物中磷酸化位点的特异性序列。我们将利用这些数据从癌细胞中挖掘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-底物关系将通过癌细胞系中相关激酶的siRNA敲低来验证。这项工作的主要目标是阐明癌细胞磷酸化网络中的关键联系。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，为激酶靶向治疗的作用机制提供见解，并为治疗干预提供新的靶点。