IDENTIFICATION OF PHOSPHORYLATION SITES ON TUMOR CELL INVASION PROTEIN

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 最近，拉希米等人。发现了一种富含脯氨酸受体的免疫球蛋白-1(IGPR-1)，它是一种参与肿瘤细胞侵袭的新的黏附受体。他们发现，依赖IGPR-1抑制细胞迁移需要SPIN90蛋白，这表明IGPR-1可能改变了SPIN90与肌动蛋白复合体的联系或其定位。SPIN90也被MAP(ERK1/ERK2)磷酸化，这增加了IGPR-1可能通过干扰SPIN90的磷酸化来抑制SPIN90的可能性。因此，他们有兴趣评估SPIN90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学是一种选择方法。对免疫沉淀样品进行消化，然后在LTQ-Orbitrap(Thermo-Fisher)上进行纳米LC-MS/MS分析。通过使用CID在SPIN90上进行的初步串联质谱学数据可以识别文献中从未报道的两个磷酸化位点和另一个已知的磷酸化位点。凝胶电泳法显示，SPIN90含有更多的磷酸化位点。因此，进一步的实验将致力于改进这一实验，以提高对磷酸化蛋白质(更多的蛋白质，不同的磷酸盐浓缩)的灵敏度。ECD或ETD将被用来保存磷酸化，便于识别磷酸化位点。为了更好地了解IGPR-1与SPIN90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.拉希米·N，马奥尼·J.E.，Gharahassanlou K.R.，Durando M.，Meyer R.D.，作为一种新的黏附受体参与肿瘤细胞侵袭的免疫球蛋白和富含脯氨酸的受体-1的鉴定，2011 2.Lim C.S.，Kim S.H.，Jung J.G.，Kim J.K.，Song W.K.，ERK对SPIN90磷酸化的调控及与NCK的相互作用，细胞黏附，J.Biol。化学，2003年，278，52,5211652123。

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.