Early Phosphorylation Kinetics of Proteins Involved in Proximal TCR-Mediated Signaling Pathways

Abstract

Activation of T cells via the stimulation of the TCR plays a central role in the adaptive immunological response. Although much is known about TCR-stimulated signaling pathways, there are still gaps in our knowledge about the kinetics and sequence of events during early activation and about the in vivo specificity of kinases involved in these proximal signaling pathways. This information is important not only for understanding the activation of signaling pathways important for T cell function but also for the development of drug targets and computer-based molecular models. In this study, phospho-specific Abs directed toward individual sites on signaling proteins were used to investigate the early phosphorylation kinetics of proteins involved in proximal TCR-induced pathways. These studies indicate that linker for activation of T cells’ tyrosines have substantially different phosphorylation kinetics and that Src homology 2 domain-containing leukocyte protein of 76 kDa has rapid, transient phosphorylation kinetics compared to other proteins. In additions, we provide evidence that ZAP-70 is the primary in vivo kinase for LAT tyrosine 191 and that Itk plays a role in the phosphorylation of tyrosine 783 on phospholipase C-γ1. In total, these studies give new insight into the sequence, kinetics and specificity of early TCR-mediated signaling events that are vital for T cell activation.

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

“…In contrast, adaptor signaling proteins such as LAT, Grb2, Gads, and SLP-76 show similar initiation timing but disappear rapidly, within 1-3 min, from these structures (61). This phenomenon was also observed by Houtman et al (62), who demonstrated that early Zap70 phosphorylation showed maximal effect of 50% at 19 s following anti-CD3 T cell stimulation, occurred maximally at 30 s, and persisted throughout the 120-s experiment. Rapid (Ͻ5 min) and strong Ag-specific phosphorylation of Zap70 has been shown to be stable for 1 h and can be reduced by nondepleting anti-CD4 Abs KT6 and YTS177 (31).…”

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK

“…SLP-76 is required for TCR-induced phosphorylation of PLC-␥1 at Y 783 (21,27). We now show that SLP-76, in particular its N-terminal tyrosine phosphorylation sites, also is required for the TCRinduced phosphorylation of the recently identified site Y 775 .…”

Differential Regulation of Human NK Cell-Mediated Cytotoxicity by the Tyrosine Kinase Itk

“…A key downstream effect of Tec kinases in hemopoietic cells is to fully activate PLC␥ contributing to a sustained release of calcium in the cell (26,27). PLC␥-dependent calcium mobilization in NK cells is required for granule exocytosis and killing.…”