TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56lck residues

Authors

  • Alice M. Nyakeriga,

    Corresponding author
    1. Department of Biomedical Sciences, Texas Tech University Health Sciences Center, 5001 El Paso Drive, El Paso, Texas 79905
    • Texas Tech University, Department of Biomedical Sciences, 5001 El Paso Dr, El Paso, TX 79905, USA
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  • Himanshu Garg,

    1. Center of Excellence of Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, 5001 El Paso Drive, El Paso, Texas 79905
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  • Anjali Joshi

    1. Center of Excellence of Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health Sciences Center, 5001 El Paso Drive, El Paso, Texas 79905
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Abstract

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56lck) 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 p56lck 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 p56lck 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 p56lck. 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 p56lck-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 p56lck residues. This implies that dephosphorylation of Y505 is not crucial for p56lck activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56lck signaling pathways in T cells at single cell level. © 2012 International Society for Advancement of Cytometry

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