The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.