Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.
Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
Sci Adv. 2019 Jun 19;5(6):eaau2001. doi: 10.1126/sciadv.aau2001. eCollection 2019 Jun.
Lck plays crucial roles in TCR signaling. We developed a new and sensitive FRET biosensor (ZapLck) to visualize Lck kinase activity with high spatiotemporal resolutions in live cells. ZapLck revealed that 62% of Lck signal was preactivated in T-cells. In Lck-deficient JCam T-cells, Lck preactivation was abolished, which can be restored to 51% by reconstitution with wild-type Lck (LckWT) but not a putatively inactive mutant LckY394F. LckWT also showed a stronger basal Lck-Lck interaction and a slower diffusion rate than LckY394F. Interestingly, aggregation of TCR receptors by antibodies in JCam cells led to a strong activation of reconstituted LckY394F similar to LckWT. Both activated LckY394F and LckWT diffused more slowly and displayed increased Lck-Lck interaction at a similar level. Therefore, these results suggest that a phosphorylatable Y394 is necessary for the basal-level interaction and preactivation of LckWT, while antibody-induced TCR aggregation can trigger the full activation of LckY394F.
Lck 在 TCR 信号中起着至关重要的作用。我们开发了一种新的、灵敏的 FRET 生物传感器（ZapLck），可在活细胞中以高时空分辨率可视化 Lck 激酶活性。ZapLck 显示，62%的 Lck 信号在 T 细胞中预先激活。在 Lck 缺陷型 JCam T 细胞中，Lck 的预先激活被消除，但可以通过用野生型 Lck（LckWT）而非假定无活性的突变体 LckY394F 重建恢复到 51%。LckWT 还显示出比 LckY394F 更强的基础 Lck-Lck 相互作用和更慢的扩散速率。有趣的是，在 JCam 细胞中，抗体诱导的 TCR 受体聚集导致重建的 LckY394F 强烈激活，类似于 LckWT。两种激活的 LckY394F 和 LckWT 的扩散速度更慢，并显示出相似水平的 Lck-Lck 相互作用增加。因此，这些结果表明，可磷酸化的 Y394 对于 LckWT 的基础水平相互作用和预先激活是必要的，而抗体诱导的 TCR 聚集可以触发 LckY394F 的完全激活。
