Phosphorylation-dependent kinetics of Zap70 activation and dissociation.

Abstract

Zap70 (“the CD3ζ-chain-associated protein kinase 70”) is one of the first proteins activated upon T cell receptor (TCR) engagement by an antigenic peptide. While it normally resides in the cytoplasm in an autoinhibited conformation, stimulation of the TCR leads to Zap70 recruitment to the phosphorylated CD3ζ chain, where Zap70 goes through phosphorylation and activation. It has been known that the full activation of Zap70 requires a multiple-step process, and growing evidence indicates that Zap70 activation can be an important kinetic proofreading step during early T cell ligand discrimination. By biochemically reconstituting the early events in the TCR signaling pathway on model lipid membranes, we probed the kinetics of Zap70 activation in a quantitative manner. A delay time between Zap70 membrane recruitment and activation was identified, and we found that both the delay time and the catalytic rate of Zap70 are regulated by its phosphorylation level. Next, we simulated the Zap70 recruitment and activation process with a stochastic model, and demonstrated that a slow activation rate is essential to reproduce the physiological Zap70 activity profile. Furthermore, through tracking individual Zap70 molecules on the TCR-decorated membrane, we found a previously unknown mechanism that extends the membrane dwell time of phosphorylated Zap70 (p-Zap70). Specifically, the dwell time profile of p-Zap70 exhibits an unexpected rise-and-fall shape, suggesting that its interaction with TCR is a nonequilibrium process. Potential functional consequences of this phosphorylation-dependent kinetic regulation in Zap70 activation and dissociation will be discussed.