TCR‐Induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56lck residues

Abstract

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56^lck) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho‐epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56^lck phosphorylation, using a multiparameter flow‐cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56^lck or cognate phospho‐tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H₂O₂ or TCR‐induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56^lck. This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56^lck‐specific inhibitor, PP2, abolished phosphorylation at either residue in a dose‐dependent manner. Taken together, these observations show that TCR‐induced stimulation of T cells led to simultaneous phosphorylation of p56^lck residues. This implies that dephosphorylation of Y505 is not crucial for p56^lck activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56^lck signaling pathways in T cells at single cell level. © 2012 International Society for Advancement of Cytometry