De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling

Abstract

The enzymatic activity of the Src family tyrosine kinase p56 (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr and Tyr Phosphorylation of Tyr and the conformational opening of Lck are believed to activate the kinase, whereas Tyr phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr plays a role in Lck function that goes beyond inducing an open conformation of the kinase.

ERK activation in CAR T cells is amplified by CD28-mediated increase in CD3ζ phosphorylation

“…These model simulations qualitatively agree with the experimental data. Similar experiments were done by Philipsen et al to test the ERK response given various LCK tyrosine to phenylalanine mutants expressed in LCK negative Jurkat T cells (Philipsen et al, 2017). They found that LCK-Y394F or LCK-Y394F-Y505F essentially eliminated the ERK positive cell population at three minutes, while LCK-Y505F increased the amount of ERK positive cells.…”

“…erning these interactions were adapted directly from our previous work . We assume that only LCK phosphorylated on the activating site, Y394, is catalytically active toward the CAR and other downstream proteins in the T cell activation pathway (Philipsen et al, 2017). As our previously published model of CAR tyrosine site phosphorylation only accounts for the catalytic activity of active LCK monophosphorylated at Y394, we assumed that doubly phosphorylated LCK, phosphorylated at the activating site Y394 and the inhibitory site Y505, has a catalytic activity 50x slower than active monophosphorylated LCK.…”

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

“…CD45 expression is required for this basal ζ-chain phosphorylation, presumably to provide for an active pool of Lck (Zikherman et al, 2010). Because a proportion of Lck is active in resting thymocytes and T cells, it has been suggested that changes in the amount of active Lck are not required to initiate TCR signaling, but these findings remain controversial (Ballek et al, 2015; Nika et al, 2010; Philipsen et al, 2017; Stirnweiss et al, 2013). However, it is known that the amount of active Lck in a T cell can determine whether it responds to antigen or not (Manz et al, 2015; Zikherman et al, 2010).…”

Selected signalling proteins recruited to the T‐cell receptor–CD3 complex

“…Different pools of Lck have been identified, including Lck in the cytoplasm, Lck anchored to the plasma membrane and Lck associated with the co‐receptors CD4 and CD8 . Approximately 40% of Lck is already active (phosphorylated at Y394) in resting T cells and upon TCR engagement the amount of active Lck increases, as seen by Forster resonance energy transfer . In addition, the distribution of Lck to its correct destination may regulate the function of Lck in phosphorylating its substrates .…”

“…29 Approximately 40% of Lck is already active (phosphorylated at Y394) in resting T cells and upon TCR engagement the amount of active Lck increases, as seen by Forster resonance energy transfer. [30][31][32] In addition, the distribution of Lck to its correct destination may regulate the function of Lck in phosphorylating its substrates. 26 Several lines of evidence have also suggested that initial phosphorylation of the CD3's ITAMs is mediated by free Lck, whereas the co-receptor-associated Lck acts as an adaptor molecule to bring the CD4 or CD8 molecule to the phosphorylated TCR-CD3 complex in a later step.…”