Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

中文翻译：

---

平行磷酸化蛋白质组学和代谢组学绘制了全球代谢酪氨酸磷酸化蛋白质组图


代谢酶的酪氨酸磷酸化是一种进化上保守的翻译后修饰，有助于酶活性、定位或功能的快速和可逆调节。尽管在基于高通量质谱的研究中，在代谢酶上检测到高丰度的酪氨酸磷酸化事件，但酪氨酸磷酸化位点的功能表征仅限于一部分酶。由于酪氨酸磷酸化在人类疾病（包括癌症）中失调，因此了解代谢酶酪氨酸磷酸化事件的后果对于为疾病生物学和治疗干预提供信息至关重要。为了全面鉴定代谢酶酪氨酸磷酸化事件并同时为这些位点分配功能意义，我们在 EGF 受体抑制剂厄洛替尼不存在的情况下，在表皮生长因子 （EGF） 刺激的非致瘤性乳腺上皮细胞 （MCF10A） 中进行了平行的磷酸化蛋白质组学和极性代谢组学。我们对磷酸化蛋白质组学和代谢组学数据集进行了综合分析，以确定代谢酶上的酪氨酸磷酸化位点及其功能后果。我们在代谢酶上确定了两个先前表征的 （丙酮酸激酶肌肉同工酶、磷酸甘油酸变位酶 1） 和两个未表征的 （谷胱甘肽 S-转移酶 Pi 1、谷氨酸脱氢酶 1） 酪氨酸磷酸化位点，这些位点具有基于代谢组学分析的据称功能。我们在 MCF10A 细胞中使用多西环素诱导的 CRISPR 干扰系统验证了这些命中，其中靶标代谢酶通过野生型、磷酸突变体或模拟磷酸化亚型的同时再表达而耗尽。 总之，这些数据为具有功能意义的代谢酶上的酪氨酸磷酸化位点的鉴定、优先级排序和表征提供了一个框架。