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J Am Chem Soc. 2018 Oct 17;140(41):13253-13259. doi: 10.1021/jacs.8b06922. Epub 2018 Oct 8.

Proteomic Identification of Protein Tyrosine Phosphatase and Substrate Interactions in Living Mammalian Cells by Genetic Encoding of Irreversible Enzyme Inhibitors.

Author information

1
State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences , Peking University , 38 Xueyuan Road , Haidian District, Beijing 100191 , China.
2
College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry , Nankai University , Tianjin 300071 , China.
3
College of Chemistry and Molecular Engineering , Peking University , Beijing 100871 , China.

Abstract

Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways, but identification of unknown PTPs for a given substrate in live cells remain technically challenging. Here, we synthesized a series of tyrosine-based irreversible PTP inhibitors and characterized by site-specific encoding on substrate proteins in cells with an expanded genetic code. By fine-tuning the chemical reactivity, we identified optimal active amino acid probes to covalently cross-link a PTP and its substrate both in vitro and in mammalian cells. Using HER2 as an example, we provide first direct evidence of HER2 Y1023 and SHP2 cross-linking in situ in living human cells. Moreover, proteomic analysis using our approach identified PTP1B as a novel phosphatase for HER2 that specifically dephosphorylated pY1221 position, which may shed light on the puzzle of PTP1B's role in HER2 positive breast cancer. This novel method provides a useful tool for dissecting tyrosine phosphoregulation in living cells.

PMID:
30247891
DOI:
10.1021/jacs.8b06922

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