Biophysical Basis Underlying Dynamic Lck Activation Visualized By Zaplck FRET Biosensor

Lymphocyte-specific protein tyrosine kinase(Lck)plays crucial roles at the initiation of T cell receptor(TCR)signals.In canonical TCR signal pathway,the first detectable molecular event is that the active Lck kinase phosphorylated the ITAM tyrosine residues in the CD3-ζ chain when TCR combined with the ligand and triggered the signal.The phosphorylated ITAM as docking site recruit Zap70 kinase and then the Zap70 was activated after phosphorylated by active Lck.Activated Zap70 involved in the TCR signal transduction cascade.However,the regulation mechanism of Lck kinase dynamics remains largely unclear during the TCR signal transduction.The kinase activity regulation of Lck depends on the phosphorylation of tyrosine 394 and tyrosine 505.In generally,the phosphorylation on tyrosine 505 inhibited the kinase activity of Lck,and its mutation leads to constitutively active of Lck.The phosphorylation on tyrosine 394 facilitates the kinase activity of Lck,and its mutation leads to the kinase-dead of Lck.However,some other study groups have different comments.Thus,the functional roles of tyrosine 394 residual in regulating Lck kinase activities are controversial.And the existing of pre-activated Lck in live cells was also controvertial in previous reports.Biochemical methods,such as western blot,were used in studying Lck kinase activity regulation,however,these classical methods could not monitor the dynamic change of Lck kinase activity in live cells.In recent years,the fluorescence(or F?ster)resonance energy transfer(FRET)methods were broadly used in the development of biosensors to monitor dynamic regulation of kinase activity in live cells.Therefore,in this project,we detected the sensitivity and specificity of series of Lck FRET biosensors,and finally developed a new and sensitive Lck FRET biosensor,Lck-FRET-Zap70FY(ZapLck),to visualize the Lck kinase activity with high spatiotemporal resolution in live T cells.We explored the dynamic changes of Lck kinase activity in TCR signal transduction process using ZapLck biosensor,and studied the mechanisms of tyrosine 394 in regulating Lck kinase activity.Here,the main results were summarized:Firstly,the characterization of ZapLck biosensor in Jurkat and Lck-deficient J.Cam1.6(JCam)T cells showed that the high specificity and sensitivity of ZapLck biosensor in monitoring activation of Lck kinase.Then the ZapLck biosensor revealing significantly pre-activated Lck in human Jurkat and PBMC T cells.Consistently,a low level of pre-activated Lck was observed and verified in Lck-deficient JCam T cells,which can be fully restored by the reconstitution of the wild-type Lck(LckWT)but not by that of a putative negative mutant,LckY394 F.Second,when co-receptors CD3 and CD28 activated by conjugated antibodies in JCam T cells expressing LckY394 F,ZapLck reported a strong activation of LckY394 F and the activation could be inhibited by adding Lck inhibitor PP1.When JCam_LckY394F cells were pre-treated with PP1,there was no detectable activation after CD3/CD28 antibody clusters stimulation.These data demonstrated that LckY394 F exerted its kinase activity involved in TCR signal transduction triggered by CD3/CD28 antibody clusters stimulation.Third,the ERK biosensor reported strong ERK kinase activation,which was downstream kinase of Lck.And strong activation was also detected in JCam_LckY394F cells indicating that LckY394 F could successfully transduce TCR signal triggered by CD3/CD28 antibody clusters to downstream ERK kinase.Furthermore,accourding to ERK biosensor monitoring,there were Lck-dependent and –independent ERK signal pathway in T cells.And the ERK could be fastly activated through Lck-dependent pathway.The results of analyzing the relationship between basal Lck expression level and its basal kinase activation in single cell revealed that the basal Lck activity in single cell is linearly proportional to the expression level of LckY394 F but non-linearly for LckWT.The FRAP experiments and analyzed results showed that the basal diffusion rate of LckY394 F was also faster than that of LckWT.After co-receptor stimulation,the diffusion rates of both LckWT and LckY394 F were reduced to similar levels,accompanied by a high activity of both LckWT and LckY394 F.Finally,to use the BiFC-split Venus system,we fused LckWT and LckY394 F with VN173 or VC155 in C-terminal.The Venus intensity analysis before and after stimulation showed that the Lck-Lck interaction was increased after stimulation both in LckWT and LckY394 F,and the LckY394 F was increased more.These results suggested that the phosphorylated tyrosine 394 in LckWT might contribute to its basal-level aggregation with slower diffusion,trans-interacting with nearby LckWT and subsequently causing its pre-activation.While LckY394 F lacks this pre-clustering and pre-activation,the antibody-mediated ligation and clustering can cause the aggregation of LckY394 F and trigger its full activation.According to all the results above,we proposed the mechanism of Lck interaction and activation.In the resting stage,the LckWT molecules form complexes via phosphorylated Y394 at T-cell membrane in a pre-activated state.Upon antigen binding,activated LckWT can be further activated and aggregate with the TCR/CD3 complex.The mutant LckY394 F molecules are relatively unbound and not pre-activated,but they can be activated and aggregate similarly to LckWT by CD3/CD28 stimulation.In summary,the new ZapLck biosensor enables the visualization of Lck kinase activation kinetics in single live T cells,unraveling the biophysical roles of Lck Y394 and its phosphorylation in mediating the intermolecular interactions of Lck for guiding kinase activities.