Shah Neel H, Wang Qi, Yan Qingrong, Karandur Deepti, Kadlecek Theresa A, Fallahee Ian R, Russ William P, Ranganathan Rama, Weiss Arthur, Kuriyan John
Department of Molecular and Cell Biology, University of California, Berkeley, United States.
California Institute for Quantitative Biosciences, University of California, Berkeley, United States.
Elife. 2016 Oct 4;5:e20105. doi: 10.7554/eLife.20105.
The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.
启动T细胞信号传导的一系列事件是由T细胞受体下游发出信号的酪氨酸激酶的激活特异性和顺序所决定的。利用一个结合了肽底物的详尽点突变、细菌表面展示、细胞分选和深度测序的平台，我们已经确定了该途径中前两个激酶Lck和ZAP-70对T细胞受体ζ链以及支架蛋白LAT和SLP-76的特异性。我们发现，ZAP-70通过利用一种静电机制来选择其底物，这种机制排除了带有带正电荷残基的底物，并有利于被带负电荷残基包围的LAT和SLP-76磷酸化位点。这种机制阻止ZAP-70磷酸化其自身的激活环，从而加强其对Lck激活的严格依赖性。ZAP-70、LAT和SLP-76中构成静电选择性基础的序列特征可能有助于T细胞对外来抗原的特异性反应。
