Paensuwan Pussadee, Hartl Frederike A, Yousefi O Sascha, Ngoenkam Jatuporn, Wipa Piyamaporn, Beck-Garcia Esmeralda, Dopfer Elaine P, Khamsri Boonruang, Sanguansermsri Donruedee, Minguet Susana, Schamel Wolfgang W, Pongcharoen Sutatip
Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany;
J Immunol. 2016 Jan 1;196(1):448-58. doi: 10.4049/jimmunol.1500958. Epub 2015 Nov 20.
Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.
配体与TCR结合会导致CD3亚基发生构象变化，从而暴露出CD3ε胞质富含脯氨酸序列（PRS）。有人提出，PRS对TCR信号传导和T细胞活化很重要。已经表明，衔接分子酪氨酸激酶非催化区域（Nck）的纯化重组SH3.1结构域可以与CD3ε暴露的PRS结合，但全长Nck在细胞中如何与TCR结合的分子机制迄今尚未得到研究。使用原位邻近连接分析和共纯化，我们表明Nck与TCR的结合需要CD3ε的部分磷酸化，因为它基于两种协同相互作用。首先，SH3.1（Nck）结构域必须与未磷酸化且暴露的PRS结合，也就是说，第一个免疫受体酪氨酸激活基序（ITAM）酪氨酸必须处于未磷酸化状态。其次，SH2（Nck）结构域必须与处于磷酸化状态的第二个ITAM酪氨酸结合。同样，Nck1中SH3.1和SH2结构域的突变导致Nck1与TCR结合丧失。此外，SH3.1突变的Nck的表达损害了TCR信号传导和T细胞活化。我们的数据表明，CD3ε磷酸化的确切模式对TCR功能至关重要。
