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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 最近，Rahimi等人。鉴定出含有富含脯氨酸的受体1（IGPR-1）的免疫球蛋白是参与肿瘤细胞侵袭的新型粘附受体。 他们发现，蛋白SPIN90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Spin90与肌动蛋白复合物或其定位的关联。 SPIN90还被MAP（ERK1/ERK2）磷酸化，从而提高了IGPR-1可以通过干扰其磷酸化来抑制SPIN90的可能性。因此，他们有兴趣在与IGPR-1相互作用后评估SPIN90上的磷酸化位点。为此，使用自下而上方法的质谱是一种选择方法。在通过LTQ-Orbitrap（Thermo-Fisher）上的Nanolc-MS/MS分析样品之前，我们对免疫沉淀样品进行了消化。使用CID在SPIN90上执行的初步串联质谱数据允许鉴定文献中从未报道过的两个磷酸化位点，并且已知的另一个位点被磷酸化。然而，凝胶电泳表明SPIN90包含更多的磷酸化位点。因此，进一步的实验将旨在改善该实验，以提高对磷酸化蛋白的敏感性（更多的蛋白质，不同的磷酸富集）。 ECD或ETD将用于保留磷酸化，从而鉴定phoshoration位点。为了更好地了解IGPR-1和SPIN90之间相互作用的机制，我们还将研究IGPR-1（糖基化，磷酸化）的翻译后修饰。 1。RahimiN.，Mahoney J.E.，Gharahassanlou K.R.，Durando M.，Meyer R.D.，鉴定含有免疫球蛋白的含有免疫球蛋白和富含脯氨酸的受体-1（IgPR-1），是涉及肿瘤细胞入侵的新型粘附受体，2011年 2。LimC.S.，Kim S.H.，Jung J.G.，Kim J.K.，Song W.K.，Spin90磷酸化和与NCK相互作用的ERK和细胞粘附的相互作用，J。Biol。 Chem。，2003，278，52，52116 52123。