Lck plays a key role in TCR signal. However, there have been very few researches of the dynamic activation regulation mechanism of Lck protein kinase in the transduction process of the signal. In addition, there has been dispute over the function and effects of tyrosine 394 residue in regulation of Lck kinase activity. The research group led by Yingxiao Wang of the University of California San Diego has collaborated with Professor Tang Liling of Chongqing University to conduct the experiment research, and has developed a new and sensitive FRET biosensor (ZapLck), which is able to display Lck kinase activity with high temporal-spatial resolution in living T cells. ZapLck has revealed that 62% of the Lck would be pre-activated in T cells. In the Lck-defect type JCam T cells, such Lck pre-activation is inhibited. After recombinant expression of wild-type Lck (LckWT), such pre-activation signal was recovered by about 51%. However, recombinant expression of non-active mutant LckY394F was not able to recover such re-activation signal. LckWT also exhibited even stronger background Lck-Lck interaction, with slower diffusion than LckY394F. Interestingly, the aggregation effect of antibody in JCam cells on TCR receptor would result in intensive activation of LckY394F, with an activation level similar to that of LckWT. The activated LckY394F and LckWT had a slow diffusion speed, which was reflected by stronger Lck-Lck interaction on the same level. As such, the above results indicate that the foundation-level interaction and pre-activation of phosphorylated Y394 on LckWT are necessary, and the TCR aggregation induced by antibody could trigger complete activation of LckY394F. The research findings were published by Science Advances, a sub-journal of Science, on line under the title “Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor” on June 19, 2019.

The research has also developed the new-type ZapLck FRET biological probe, which is able to monitor the change of activation kinetics of Lck kinase in a single living T cell in a specific manner. In addition, the research has revealed the biological and physical effect of LckY394F and the mechanism of tyrosine 394-residue in mediating interactions between Lck molecules and directing phosphorylation of activation of kinase. The ZapLck biological probe serves as a powerful tool for Lck related researches, and the research findings could be used as new theoretical basis for study of Lck function and TCR signal transduction events.

This research has been completed as directed by Professor Yingxiao Wang and Shaoying Lu from UC San Diego, with Wan Rongxue, a doctoral candidate of Chongqing University, as the first author. Professor Yingxiao Wang, Professor Shaoying Lu and Professor Tang Liling are corresponding co-authors.