A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

Protein Tyrosine Phosphatase Non-receptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 post-translational regulation. Here we characterize a phosphorylation site at Ser325 situated C-terminal to the catalytic domain of PTPN22, and its roles in altering protein function. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9 mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Next-generation sequencing (NGS) revealed it differentially regulates the expression of chemokines and T cell activation pathways. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling . We generated the CRISPR/Cas9 mediated mutagenesis of Ser325 to glutamic acid and Alanine to explore the roles of Ser325 phosphorylation in activated Jurkat T cells The cells were starved and stimulated by antibodies against human CD3/CD28 antibodies for 16h, and cells were collected, washed and used for bulk RNA sequencing.

非受体型蛋白酪氨酸磷酸酶22（Protein Tyrosine Phosphatase Non-receptor type 22，PTPN22）由核心自身免疫相关基因编码，是公认的T细胞受体（TCR）信号通路抑制剂，同时也是癌症免疫治疗的药物靶点。然而，目前对于PTPN22的翻译后调控机制仍知之甚少。本研究对PTPN22催化结构域C端的Ser325磷酸化位点及其对蛋白质功能的调控作用进行了表征。通过CRISPR/Cas9介导抑制Ser325磷酸化可增强PTPN22调控的核心TCR依赖型信号通路活性，而通过谷氨酸（glutamic acid）替换模拟该磷酸化状态则会抑制该通路。下一代测序（Next-generation sequencing，NGS）结果显示，该位点可差异性调控趋化因子及T细胞活化通路的基因表达。综上，本研究阐明了PTPN22的一个新型磷酸化位点的功能，该位点参与TCR信号通路的复杂调控。为探究Ser325磷酸化在活化Jurkat T细胞中的作用，我们通过CRISPR/Cas9介导构建了Ser325谷氨酸替换及丙氨酸（Alanine）突变的细胞模型。将细胞进行血清饥饿处理后，使用抗人CD3/CD28抗体刺激16小时，随后收集、洗涤细胞并进行批量RNA测序（bulk RNA sequencing）。