Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22128-33. doi: 10.1073/pnas.1016388108. Epub 2010 Dec 6.
Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/CD3 signaling after the receptor internalization.
T 细胞受体复合物 (TcR/CD3) 的磷酸化介导 T 细胞的存活和抗原诱导的激活。通常使用磷酸化特异性抗体来监测 TcR/CD3 的磷酸化，这排除了动态测量。在这里，我们开发了遗传编码的活细胞报告器，能够同时监测 CD3ζ 的磷酸化状态和细胞内转运，CD3ζ 是 TcR/CD3 的主要信号转导亚基。我们表明，这些报告器提供了 TcR/CD3 磷酸化的准确读数，并且对作用于 TcR/CD3 的激酶和磷酸酶活性的局部平衡敏感。使用这些报告器，我们证明，除了在质膜上预期的激活依赖性磷酸化之外，酪氨酸磷酸化的 CD3ζ 在与溶酶体不同的内体小泡上积累。这些结果表明，磷酸化的 CD3ζ 的细胞内池可能有助于在受体内化后维持 TcR/CD3 信号转导。
