Proteomic Identification of Protein Tyrosine Phosphatase and Substrate Interactions in Living Mammalian Cells by Genetic Encoding of Irreversible Enzyme Inhibitors
Proteomic Identification of Protein Tyrosine Phosphatase and Substrate Interactions in Living Mammalian Cells by Genetic Encoding of Irreversible Enzyme Inhibitors
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通过不可逆酶抑制剂的基因编码对活体哺乳动物细胞中蛋白质酪氨酸磷酸酶和底物相互作用进行蛋白质组学鉴定
DOI：
10.1021/jacs.8b06922
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发表时间：
2018
影响因子：
15
通讯作者：
Liu Tao
中科院分区：
文献类型：
--
作者：
Tang Hongting;Dai Zhen;Qin Xuewen;Cai Wenkang;Hu Liming;Huang Yujia;Cao Wenbing;Yang Fan;Wang Chu;Liu Tao
蛋白质酪氨酸磷酸酶(PTPs)在细胞信号通路中起着关键作用，但在活细胞中识别特定底物的未知PTPs在技术上仍然具有挑战性。在这里，我们合成了一系列基于酪氨酸的不可逆的PTP抑制剂，并在具有扩展遗传密码的细胞中对底物蛋白进行了定点编码。通过微调化学反应活性，我们找到了在体外和哺乳动物细胞中以共价方式交联PTP及其底物的最佳活性氨基酸探针。以HER2为例，我们首次提供了HER2 Y1023和SHP2在活体细胞中原位交联的直接证据。此外，利用我们的方法进行的蛋白质组学分析表明，PTP1B是一种新的HER2磷酸酶，它特异性地去磷酸化了pY1221位，这可能有助于解开PTP1B在HER2阳性乳腺癌中所扮演的角色的谜团。这一新的方法为分析活细胞中的酪氨酸磷酸调节提供了一个有用的工具。
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.