Leibniz Institute of Molecular Pharmacology (FMP), Robert Roessle Strasse 10, 13125 Berlin, Germany.
J Am Chem Soc. 2010 Oct 27;132(42):14704-5. doi: 10.1021/ja106764y.
Post-translational protein modifications (PTMs) such as phosphorylation and acetylation regulate a large number of eukaryotic signaling processes. In most instances, it is the combination of different PTMs that "encode" the biological outcome of these covalent amendments in a highly dynamic and cell-state-specific manner. Most research tools fail to detect different PTMs in a single experiment and are unable to directly observe dynamic PTM states in complex environments such as cell extracts or intact cells. Here we describe in situ observations of phosphorylation and acetylation reactions by high-resolution liquid-state NMR spectroscopy. We delineate the NMR characteristics of progressive lysine acetylation and provide in vitro examples of joint phosphorylation and acetylation events and how they can be deciphered on a residue-specific basis and in a time-resolved and quantitative manner. Finally, we extend our NMR investigations to cellular phosphorylation and acetylation events in human cell extracts and demonstrate the unique ability of NMR spectroscopy to simultaneously report the establishment of these PTMs by endogenous cellular enzymes.
翻译后蛋白质修饰（PTM），如磷酸化和乙酰化，调节大量真核生物信号传导过程。在大多数情况下，正是不同PTM的组合以高度动态且细胞状态特异性的方式“编码”了这些共价修饰的生物学结果。大多数研究工具无法在单个实验中检测到不同的PTM，并且无法在复杂环境（如细胞提取物或完整细胞）中直接观察动态PTM状态。在此，我们描述了通过高分辨率液态核磁共振光谱对磷酸化和乙酰化反应的原位观察。我们描绘了赖氨酸渐进性乙酰化的核磁共振特征，并提供了联合磷酸化和乙酰化事件的体外实例，以及如何在残基特异性基础上、以时间分辨和定量的方式对它们进行解读。最后，我们将核磁共振研究扩展到人类细胞提取物中的细胞磷酸化和乙酰化事件，并证明了核磁共振光谱能够独特地报告内源性细胞酶建立这些PTM的情况。
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