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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cross-Linking Reagents / chemical synthesis
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Cross-Linking Reagents / chemistry*
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Cysteine / chemistry
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Enzyme Inhibitors / chemical synthesis
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Enzyme Inhibitors / chemistry*
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HEK293 Cells
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Humans
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Phosphorylation / physiology
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Proof of Concept Study
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Protein Engineering / methods
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Protein Tyrosine Phosphatase, Non-Receptor Type 1 / analysis*
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Protein Tyrosine Phosphatase, Non-Receptor Type 1 / antagonists & inhibitors
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Protein Tyrosine Phosphatase, Non-Receptor Type 1 / chemistry
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Protein Tyrosine Phosphatase, Non-Receptor Type 1 / metabolism
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / chemistry
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Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism
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Proteomics / methods
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Receptor, ErbB-2 / chemistry
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Receptor, ErbB-2 / genetics*
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Receptor, ErbB-2 / metabolism*
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Tyrosine / analogs & derivatives
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Tyrosine / chemical synthesis
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Tyrosine / genetics*
Substances
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Cross-Linking Reagents
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Enzyme Inhibitors
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Tyrosine
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ERBB2 protein, human
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Receptor, ErbB-2
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PTPN1 protein, human
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Protein Tyrosine Phosphatase, Non-Receptor Type 1
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Cysteine