IDENTIFICATION OF PHOSPHORYLATION SITES ON TUMOR CELL INVASION PROTEIN

肿瘤细胞侵袭蛋白磷酸化位点的鉴定

• 批准号: 8365581
• 负责人: Nader Rahimi
• 金额: $ 0.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365581
• 关键词: Actins; Biology; Cell Adhesion; Cell Migration Inhibition function; Complex; Data; Digestion; Funding; Grant; Immune; Immunoglobulins; Literature; MAPK1 gene; MAPK3 gene; Mass Spectrum Analysis; Medicine; Methods; National Center for Research Resources; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Principal Investigator; Proline; Proteins; Regulation; Reporting; Research; Research Infrastructure; Resources; SHFM1 gene; Sampling; Site; Source; Tumor Cell Invasion; United States National Institutes of Health; adhesion receptor; cost; gel electrophoresis; glycosylation; improved; inorganic phosphate; interest; novel; receptor; research study; tandem mass spectrometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recently, Rahimi et al. identified an immunoglobulin containing proline rich receptor-1 (IGPR-1) as a novel adhesion receptor involved in tumor cell invasion. They found that the protein SPIN90 is required for IGPR-1 dependent inhibition of cell migration, suggesting that perhaps IGPR-1 alters association of SPIN90 with actin complexes or its localization. SPIN90 also has been shown to be phosphorylated by MAP (ERK1/ERK2), raising a possibility that IGPR-1 may inhibit SPIN90 by interfering with its phosphorylation. Hence, they are interested to evaluate the phosphorylation sites on the SPIN90 after its interaction with IGPR-1. For this purpose, mass spectrometry using bottom-up approach is a method of choice. We performed digestion on the immune-precipitated samples, before analyzing samples by nanoLC-MS/MS on LTQ-Orbitrap (Thermo-Fisher). Preliminary tandem mass spectrometry data performed on SPIN90 by using CID allowed identification of two phosphorylation sites never reported in the literature and another site known to be phosphorylated. However, gel electrophoresis suggested that SPIN90 contained more phosphorylation sites. Hence, further experiments will aim to improve this experiment to increase the sensitivity for the phosphorylated protein (more protein, different phosphate enrichment). ECD or ETD will be used to preserve the phosphorylation, facilitying the identification of the phoshorylation site. To have a better understanding of the mechanism of the interaction between the IGPR-1 and SPIN90, we will also investigate the post-translational modifications on the IGPR-1 (glycosylation, phosphorylation). 1. Rahimi N., Mahoney J.E., Gharahassanlou K.R., Durando M., Meyer R.D., Identification of immunoglobulin containing and proline rich receptor-1 (IGPR-1) as a novel adhesion receptor involved in tumor cell invasion, 2011 2. Lim C.S., Kim S.H., Jung J.G., Kim J.K., Song W.K., Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion, J. Biol. Chem., 2003, 278, 52, 5211652123.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 最近，Rahimi等鉴定了含有富含脯氨酸的受体-1（IGPR-1）的免疫球蛋白作为参与肿瘤细胞侵袭的新型粘附受体。 他们发现，蛋白质SPIN 90是IGPR-1依赖性抑制细胞迁移所必需的，这表明IGPR-1可能改变了SPIN 90与肌动蛋白复合物的结合或其定位。SPIN 90也被MAP磷酸化（ERK 1/ERK 2），这增加了IGPR-1可能通过干扰其磷酸化来抑制SPIN 90的可能性。因此，他们有兴趣评估SPIN 90与IGPR-1相互作用后的磷酸化位点。为此，使用自下而上方法的质谱法是一种选择方法。我们对免疫沉淀的样品进行消化，然后在LTQ-Orbitrap（Thermo-Fisher）上通过nanoLC-MS/MS分析样品。通过使用CID对SPIN 90进行的初步串联质谱数据允许鉴定文献中从未报道的两个磷酸化位点和另一个已知被磷酸化的位点。凝胶电泳结果显示SPIN 90含有更多的磷酸化位点。因此，进一步的实验将旨在改进该实验以增加对磷酸化蛋白质的灵敏度（更多蛋白质，不同的磷酸盐富集）。ECD或ETD将用于保存磷酸化，便于鉴定磷酸化位点。为了更好地理解IGPR-1和SPIN 90之间相互作用的机制，我们还将研究IGPR-1的翻译后修饰（糖基化，磷酸化）。 1. Rahimi N.，马奥尼J.E.，Gharahassanlou K.R.，Durando M.，迈耶研究所，鉴定含有免疫球蛋白和富含脯氨酸的受体-1（IGPR-1）作为参与肿瘤细胞侵袭的新型粘附受体，2011年 2. Lim C.S.，Kim S.H.，Jung J.G.，Kim J.K.，宋伟光，ERK和细胞粘附对SPIN 90磷酸化的调节以及与Nck的相互作用，生物化学杂志，2003，278，52，5211652123.