Layered post-translational modifications provide a tuning method for ubiquitylation

项目总结/摘要 蛋白质磷酸化是一种重要的翻译后修饰，几乎调节细胞的各个方面 生物学异常磷酸化驱动致癌过程，如不受控制的细胞增殖，细胞迁移， 和更多.磷酸化最近被证明赋予了一个额外的调控层， 泛素化，另一个重要的调节翻译后修饰。该提案的目标是发展 用于研究和绘制癌组织中异常蛋白质磷酸化模式的化学工具; 具体地说，我们提出了一种方法，发现负责磷酸化泛素的激酶， 癌组织我们观察到磷酸化位点位于关键结构基序中，这些基序是 泛素与其效应物相互作用，并且在某些位点的磷酸化已经被观察到， 肿瘤样本因此，我们假设泛素磷酸化调节泛素结构， 修饰其与效应蛋白的相互作用、分支模式和分布， 磷酸化位点可能是病理性的。因为目前的方法不能回答哪些或多少激酶 能够使磷酸化位点磷酸化，目的1提出优化化学交联剂， 与给定磷酸化位点相互作用的激酶的特异性共价捕获。该系统呈现 几个理论上的优势，即它可以观察到多种激酶的简并磷酸化， 在天然浓度的激酶和存在天然效应物的情况下评估交联。 初步数据表明，我们已经开发出一种共价交联剂，能够交联激酶， 半胱氨酸突变蛋白底物和能够将激酶连接至修饰的半胱氨酸突变蛋白底物的生物正交交联剂 底物肽。在目标2中，我们建议将这种交联剂应用于泛素磷酸化的研究， 四个部位，在生理和病理条件下确定。该提案的完成将提供新的 化学工具，以解决激酶生物学以前未探索的方面，并将阐明 泛素是一种在癌症中失调的必需调节蛋白。

Project Summary / Abstract Protein phosphorylation is an essential posttranslational modification regulating nearly every aspect of cell biology. Aberrant phosphorylation drives oncogenic processes like uncontrolled cell proliferation, cell migration, and more. Phosphorylation was recently shown to confer an additional layer of regulatory control over ubiquitylation, another critical regulatory posttranslational modification. The goal of this proposal is to develop chemical tools for studying and mapping patterns of aberrant protein phosphorylation in cancerous tissue; specifically, we propose a method to discover the kinases responsible for phosphorylating ubiquitin in cancerous tissue. We observe that phosphorylation sites are located in key structural motifs necessary for ubiquitin to interact with its effectors and that phosphorylation at some sites has been observed exclusively in tumor samples. As such, we hypothesize that ubiquitin phosphorylation modulates ubiquitin structure, modifying its interaction with effector proteins, branching patterns and distribution, and that some phosphorylation sites may be pathologic. Because current methods cannot answer which or how many kinases are capable of phosphorylating a phosphorylation site, Aim 1 proposes to optimize a chemical crosslinker for the specific covalent capture of kinases which interact with a given phosphorylation site. This system presents several theoretical advantages; namely, it could observe degenerate phosphorylation by multiple kinases and crosslinking would be assessed under native concentrations of kinase and with native effectors present. Preliminary data show that we have developed a covalent crosslinker capable of crosslinking kinases to their cysteine mutant protein substrates and a bioorthogonal crosslinker capable of ligating kinases to modified substrate peptide. In Aim 2, we propose to apply this crosslinker to the study of ubiquitin phosphorylation at four sites, identified under physiologic and pathologic conditions. Completion of this proposal will provide a new chemical tool to address a previously unexplored aspect of kinase biology and will shed light on the posttranslational modification of ubiquitin, an essential regulatory protein that is dysregulated in cancer.