T cells are activated when their T cell receptors (TCR) interact with antigens in the form of agonist peptides presented in the context of a major histocompatibility complex (MHC). T cells must remain quiescent when presented with highly abundant low affinity self peptide MHC interactions. To achieve this it is generally accepted that a kinetic proof-reading mechanism exists to filter out noise induced by short-lived binding events, but the molecular mechanism remains ill-defined. Zeta-chain-associated protein kinase 70 (ZAP70) plays a critical role in the proximal signaling cascade downstream of T cell receptor activation, where it is recruited to phosphorylated immunotyrosine-based activation motifs (ITAMs) in the TCR. ZAP70 contains tandem Src homology 2 (SH2) domains that bind with high specificity and affinity to the two phospho-tyrosines in ITAMs, but such stable interactions would be incompatible with a role in the kinetic proof-reading mechanism. To mediate this role the duration of ZAP70 recruitment to pMHC-engaged TCR should depend on the lifetime of the TCR-pMHC interaction.
Using total internal reflection microscopy and single particle tracking we assessed the lifetime of a ZAP70 at the membrane in ILA TCR-expressing Jurkat T cells activated with four different altered peptide ligands of differing affinities. As our hypothesis suggests the membrane lifetime of individual ZAP70 molecules correlated with the lifetime of TCR-pMHC interactions. We next directly measured the binding kinetics of ZAP70 on phosphorylated ITAMs using surface plasmon resonance and microscopy. The results of kinetic measurements on mono- and bi-phosphorylated ITAMs support a model in which ZAP70 rapidly cycles between monovalent and bivalent binding states. In monovalent states one tyrosine is available for dephosphorylation by phosphatases, which would prevent ZAP70 from cycling into the bivalent conformation and thus shorten the overall complex lifetime. In support of this model inclusion of a protein tyrosine phosphatase accelerated ZAP70-ITAM dissociation. Taken together, these results indicate that tandem SH2 domains allow bond lifetimes with ITAMs to be regulated by the local activity of phosphatases in a manner that a monovalent interaction would not be. We propose that this allows ZAP70 membrane recruitment to be sensitive to TCR-pMHC binding lifetimes and thus allows it to function as a step in the kinetic proofreading mechanism. Given the prevalence of proteins with multiple SH2 domains this mechanism may function generally in many signalling reactions that require rapid kinetic tuning that is sensitive to the local balance of kinase and phosphatase activities.