Kinetic Analysis of ZAP70 Phosphorylation in Human T cells: p-Y292, p-Y319, and p-Y493

Abstract

Abstract The 70-kDa zeta chain-associated protein (ZAP-70) is a Syk family protein tyrosine kinase normally expressed in T and NK cells. Patients deficient in ZAP-70 have no functional T cells in the periphery, are prone to repeated infections, and suffer from ZAP-70-related severe combined immunodeficiency (SCID). In some cases of chronic lymphocytic leukemia (CLL), ZAP-70 is aberrantly expressed in B cells and used a prognostic marker to monitor disease progression and therapeutic intervention. In normal T cells, ZAP-70 is required for thymic development, T cell receptor (TCR) mediated signaling, and T cell activation. ZAP-70 consists of two tandem Src homology 2 (SH2) domains, two linker regions, and a kinase domain. Following TCR engagement, several tyrosine residues within ZAP-70 are phosphorylated and serve to both positively and negatively regulate kinase activity. A kinetic analysis was performed using human peripheral blood mononuclear cells (PMBCs) stimulated with hydrogen peroxide (H2O2), anti-CD3/CD28, anti-CD3/PD-1, or anti-CD3/TIGIT. Using in-house generated clones, T cells were analyzed for three distinct ZAP-70 phospho-epitopes: p-Y292, p-Y319, and p-Y493.