Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

• 成果类型: Article
• 署名作者: Hillis, Alissandra L.; Tamir, Tigist; Perry, Grace E.; Asara, John M.; Johnson, Jared L.; Yaron, Tomer M.; Cantley, Lewis C.; White, Forest M.; Toker, Alex
• 署名单位: Harvard University; Harvard University Medical Affiliates; Beth Israel Deaconess Medical Center; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Beth Israel Deaconess Medical Center; Harvard Medical School; Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); Harvard University; Harvard Medical School; Harvard University Medical Affiliates; Beth Israel Deaconess Medical Center; Harvard University; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Dana-Farber Cancer Institute; Harvard Medical School; Cornell University; Weill Cornell Medicine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14129
• DOI: 10.1073/pnas.2413837121
• 发表日期: 2024-11-19
• 关键词: s-transferase p1; phosphorylation; phosphatases; mutations; isoform; kinases

摘要

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.