Research ArticleImmunology

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

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Sci. Signal.  17 Jan 2017:
Vol. 10, Issue 462, eaaf4736
DOI: 10.1126/scisignal.aaf4736

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Lck: When an open conformation does not equal activation

The tyrosine kinase Lck is critical to T cell activation in response to stimulation of the T cell receptor (TCR). Lck activity is tightly regulated to avoid inappropriate activation of T cells and subsequent inflammation. Phosphorylation of Tyr505 causes Lck to form a closed, inhibited conformation, whereas phosphorylation of Tyr394 results in conformational opening of the kinase. To tease apart the differential effects of phosphorylation and conformational changes on Lck activity, Philipsen et al. generated different fluorescent Lck biosensors and imaged unstimulated and TCR-stimulated human T cells by fluorescence microscopy. Both the TCR-stimulated conformational opening of Lck and its subsequent phosphorylation of Tyr394 were required to stimulate T cells, and these modifications occurred primarily on Lck that was close to that TCR at the plasma membrane. These data suggest that drugs that stabilize the open conformation yet prevent the phosphorylation event could limit T cell–mediated immune responses.

Abstract

The enzymatic activity of the Src family tyrosine kinase p56Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394 and Tyr505. Phosphorylation of Tyr394 and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505 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 Tyr394 to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394 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 Tyr394 plays a role in Lck function that goes beyond inducing an open conformation of the kinase.

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