ζ Phosphorylation Without ZAP-70 Activation Induced by TCR Antagonists or Partial Agonists

Abstract

Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling

“…In contrast, the Th2 cells showed less overall TCR-associated protein tyrosine phosphorylation and predominant generation of partially phosphorylated (p21) TCR f chain in combination with nearly undetectable phosphorylation of ZAP-70. This latter pattern corresponds to that observed using partial agonists or antagonists to stimulate naive or Th1-polarized CD4 + or CD8 + T cells [17,18]. It also resembles the signaling pattern seen with Th1 cells stimulated under conditions in which CD4-MHC class II interactions are disrupted [14], suggesting a possible role for reduced CD4 function in the signaling behavior of Th2 cells.…”

“…2). The Th1 cells showed phosphorylation of ZAP-70 and a nearly equal generation of the two main forms of phospho-f (p21 and p23), corresponding to the full activation pattern induced by agonist ligands as reported in previous studies of naive or Th1-polarized T cells [17,18]. In contrast, the Th2 cells showed less overall TCR-associated protein tyrosine phosphorylation and predominant generation of partially phosphorylated (p21) TCR f chain in combination with nearly undetectable phosphorylation of ZAP-70.…”

“…After incubation, cells were lysed with 1% Nonidet-P40, 140 mM NaCl, 10 mM Tris-HCl (pH 7.2), 2 mM EDTA, 5 mM iodoacetamide, 1 mM Na 3 VO 4 , and proteinase inhibitor mixture (Boehringer Mannheim, Indianapolis, IN) on ice for 30 min. After centrifugation, the supernatant was incubated with anti-TCR (clone H57-597), biotinylated anti-CD3e (clone 145-2C11), or antiserum to ZAP-70 ( [17], kindly provided by Dr. J. B. Bolen) for 4 h and precipitated with protein A-Sepharose (Amersham Pharmacia Biotech, Sweden) or ImmunoPure immobilized streptavidin (Pierce, Rockford, IL) for biotinylated antibody.…”

“…Horseradish peroxidase activity was detected with ECL (Amersham Pharmacia Biotech). Previous studies have shown that these solubilization and precipitation conditions are appropriate for the detection of phosphoproteins associated with the proximal events in TCR-dependent signaling [14,15,[17][18][19]. Controls for sample loading consisted of re-probing of blots for total ZAP-70.…”

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

“…These experiments indicate that ZAP-70 association with the TCR/CD3 is dependent on signals that are provided by Lck and/or Fyn (23,24), supporting the role of Lck and/or Fyn in early tyrosine phosphorylation of ITAMs within the TCR/CD3 subunits. ZAP-70 recruitment to the TCR via binding of the tandem SH2 domains of ZAP-70 to tyrosine phosphorylated ITAMs is a prerequisite for its activation and elicitation of subsequent biochemical events (25,26), although the precise role of recruitment in activating ZAP-70 remains unclear. It is also yet unknown which of the proteins * The costs of publication of this article were defrayed in part by the payment of page charges.…”