Layered post-translational modifications provide a tuning method for ubiquitylation

项目摘要/摘要 蛋白质磷酸化是一种基本的翻译后修饰，几乎调节细胞的各个方面 生物学。异常的磷酸化推动了致癌过程，如失控的细胞增殖、细胞迁移、 还有更多。最近的研究表明，磷酸化可以提供一层额外的调控 泛素化，另一个关键的调控翻译后修饰。这项提议的目标是发展 研究和绘制癌症组织中异常蛋白磷酸化模式的化学工具； 具体地说，我们提出了一种方法来发现负责磷酸化泛素的激酶。 癌变组织。我们观察到，磷酸化位点位于关键的结构基序中，这是 泛素与其效应器相互作用，并且某些部位的磷酸化仅在 肿瘤样本。因此，我们假设泛素磷酸化调节泛素结构， 改变其与效应蛋白的相互作用、分支模式和分布，以及一些 磷酸化位点可能是病理性的。因为目前的方法不能回答是哪种或有多少个激酶 能够使磷酸化位点磷酸化，目标1建议优化一种化学交联剂 与给定的磷酸化位点相互作用的激酶的特定共价捕获。该系统提供了 几个理论上的优势；即，它可以观察到多个激酶和 交联性将在天然浓度的激酶和天然效应物存在的情况下进行评估。 初步数据显示，我们已经开发出一种共价交联剂，能够将激酶与它们的 半胱氨酸突变蛋白底物和生物正交联剂 底物多肽。在目标2中，我们建议将这种交联剂用于泛素磷酸化的研究。 四个部位，在生理和病理条件下鉴定。这项提案的完成将提供一个新的 一种化学工具，用于解决以前未被探索的激酶生物学方面，并将阐明 泛素的翻译后修饰，泛素是一种在癌症中调节失调的重要调节蛋白。

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.