TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56(lck)  residues.
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 H(2) O(2) 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.
Department of Biomedical Sciences, Texas Tech University Health Sciences Center, 5001 El Paso Drive, El Paso, Texas 79905, USA. alice.nyakeriga@ttuhsc.edu
Alice M
AM
Nyakeriga
Himanshu
H
Garg
Anjali
A
Joshi
D000293:Adolescent; D000328:Adult; D020816:Amino Acid Motifs; D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism; D020013:Calcium Signaling; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000378:metabolism; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008875:Middle Aged; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011743:Pyrimidines / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D055815:Young Adult; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D023201:CD40 Ligand; D006861:Hydrogen Peroxide; D037181:Lectins, C-Type; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D011743:Pyrimidines; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D000099192:CD69 Antigens; C412373:AG 1879