酪氨酸磷酸酶SHP1新型作用底物THEMIS的鉴定及其在免疫T细胞发育过程中的功能探究

酪氨酸磷酸酶家族，协同酪氨酸激酶，共同参与调节细胞的各项生命活动。而整个酪氨酸磷酸酶研究领域的痛点在于系统性地发掘受其直接调控的作用底物。基于此，我们将酪氨酸磷酸酶特有的底物捕获技术和最新开发的邻近标记技术相结合，构建出可以实现高通量操作的酪氨酸磷酸酶底物鉴定平台（PEPSI）。我们将平台运用到免疫细胞中一个重要的酪氨酸磷酸酶基因SHP1上，成功鉴定出其在T细胞发育以及激活过程中参与调节的全新底物THEMIS，并通过生化实验验证了THEMIS特定位点的磷酸化以及去磷酸化的确受到激酶LCK和磷酸酶SHP1的调控。我们希望依托面上项目的资助完善PEPSI平台，并通过建立体外细胞系统以及体内转基因小鼠系统，进一步研究THEMIS上该特定磷酸化位点对于T细胞信号转导的重要作用。鉴于SHP1在肿瘤免疫中的拮抗作用，我们的研究成果也将对其作为免疫检查点抑制剂协同靶点的可能性提供关键的理论依据。

Reversible tyrosine phosphorylation, which fine-tuned by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical for cells to recognize and response to stimuli in their environment. The ability to define the substrate specificity of members of the PTP family is an essential aspect of gaining an understanding of the function of these enzymes. By combining substrate trapping and pupylation-based interaction tagging (PUP-IT) strategies, we here developed a systematic proximity labeling technology PEPSI (PUP-IT Enhanced PTP Substrate Identification) for identifying novel de-phosphorylation substrates of protein tyrosine phosphatases. We applied the method with human PTPN6/SHP1, and thereby identified THEMIS as a previously unknown substrate of this well-known T cell activation regulating phosphatase. Our preliminary studies have demonstrated that a phosphorylation modification of the THEMIS tyrosine residue—added by LCK and removed by PTPN6/SHP1—is essential for T cell activation. With this great funding opportunity from NSFC, we will further study the function of this particular tyrosine phosphorylation on THEMIS in the context of TCR signaling transduction and T cell development in vivo. Because of the negative regulatory role of SHP1 in tumor immunotherapy, our research will shed more insight on the synergistic effect of co-targeting this important PTP in immune cells along with immune checkpoint blockade.