FRET biosensor to visual Lck kinase activity during TCR activation

Author(s): Wu, Jenny | Advisor(s): Wang, Yingxiao | Abstract: On the molecular level, the immune response begins in T-cells where lymphocyte-specific protein tyrosine kinase p56­Lck (Lck), a Src family kinase (SFK), is one of the first molecules involved in early T-cell activation at T-cell receptors (TCRs). A biosensor utilizing fluorescence resonance energy transfer (FRET) was designed to better understand and monitor Lck kinase activity during TCR activation by undergoing FRET change when an active Lck phosphorylates the biosensor. According to the in vitro data, the Lck FRET biosensor can also report proto-oncogene tyrosine-protein kinase p59­Fyn (Fyn) activity faster than to Lck activity, implying that the Fyn kinase has a higher activity level than the Lck kinase. However, mammalian HeLa cell data showed that the biosensor is more specific to Lck in a physiological setting where kinase-dead Lck(K273R) and Lck(Y394F) can induce a higher FRET change than Lck(WT). In J.CaM1.6 cells, Lck-deficit T-cells, kinase-dead Lck(K273R) did not elicit a FRET change, but kinase-dead Lck(Y394F) continued to elicit a higher biosensor response than Lck(WT). This suggests that Lck may have an adaptor function to recruit other Src family kinases (SFKs) for continuous phosphorylation on the biosensor.